US20030109067A1 - Homogeneous immunoassays for multiple allergens - Google Patents

Homogeneous immunoassays for multiple allergens Download PDF

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Publication number
US20030109067A1
US20030109067A1 US10/006,483 US648301A US2003109067A1 US 20030109067 A1 US20030109067 A1 US 20030109067A1 US 648301 A US648301 A US 648301A US 2003109067 A1 US2003109067 A1 US 2003109067A1
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conjugate
antibody
binding pair
particles
specific binding
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Christopher Brown
James Murai
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Immunetech Inc
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Immunetech Inc
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Priority to US10/006,483 priority Critical patent/US20030109067A1/en
Application filed by Immunetech Inc filed Critical Immunetech Inc
Priority to DK02786929.6T priority patent/DK1461618T3/da
Priority to PCT/US2002/039111 priority patent/WO2003050539A1/en
Priority to ES02786929T priority patent/ES2340667T3/es
Priority to AU2002351281A priority patent/AU2002351281A1/en
Priority to EP02786929A priority patent/EP1461618B1/en
Priority to AT02786929T priority patent/ATE457459T1/de
Priority to DE60235324T priority patent/DE60235324D1/de
Priority to JP2003551541A priority patent/JP2005512091A/ja
Priority to US10/387,790 priority patent/US20030232397A1/en
Publication of US20030109067A1 publication Critical patent/US20030109067A1/en
Priority to US11/786,193 priority patent/US7491553B2/en
Priority to US12/316,838 priority patent/US7892853B2/en
Priority to US13/006,394 priority patent/US8034632B2/en
Priority to US13/006,392 priority patent/US8034631B2/en
Priority to US13/006,398 priority patent/US8093066B2/en
Priority to US13/312,977 priority patent/US8349620B2/en
Priority to US13/335,808 priority patent/US20120264230A1/en
Abandoned legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • This invention relates to a homogenous immunoassay method for determining specific antibody levels to a multiplicity of allergens from a blood sample, or for determining total immunoglobulins E levels in such a sample, for the purpose of diagnosing allergy.
  • allergens are synonymous with atopy or hypersensitivity and is the result of an immunologically mediated reaction by individuals to various antigenic materials, known as allergens. People with allergies produce allergen-specific immunoglobulins, IgE and IgG, in response to exposure to normally harmless substances from pollens, molds, dander or foods, which are inhaled or ingested. The generated antibodies are released to circulate in the blood and eventually fix to specific cells in tissue. Exposure to allergens generally results in immediate or delayed reactions, manifested in a number of commonly identifiable symptoms, such as sneezing, itchy eyes, runny nose and inflammation of the lungs and nasal passages.
  • allergy is also generally synonymous with hay fever, rhinitis, eczema, hives, and linked to the onset of asthma.
  • the diagnosis of allergy involves a review of the patient history, physical examinations and running a confirmatory diagnostic test to identify whether the patient's symptoms are of allergic or non-allergic origin. If allergy is responsible for the symptoms, then the allergens responsible must be identified. Patients with atopic or allergic diseases may be mono-sensitive to one allergen; however, sensitization to multiple allergens is more usual. Reactions of persons to allergens can range from the annoying to the severe or even fatal. It therefore is desirable to be able to determine not only whether a person has allergies, but if so, to what allergens and to what level of severity, so that exposure can be avoided, minimized or mitigated through pharmacotherapeutic or immunotherapeutic methods
  • Confirmatory diagnostic testing may be conducted by in-vivo skin testing, in-vivo provocation testing, or in-vitro testing for the presence of circulating allergen-specific antibodies from blood samples.
  • Direct provocation by direct inhalation or ingestion of possible offending allergens, while relevant, is unpleasant, possibly dangerous and cannot be performed for multiple allergens at one sitting.
  • Skin testing is an in vivo procedure that involves applying an allergen sample, or more generally a multiplicity of allergens, directly to a patient's forearm or back via a small needle scratch and measuring the size of the inflammatory reaction (wheal) at the applied site on the skin.
  • Skin prick testing is widely used, is reliable under optimal testing conditions, can be painful, is subject to large differences in technique and interpretations, and cannot be used on patients taking certain drugs or patients with skin problems.
  • both provocation and skin prick in-vivo diagnostic methods have the potential for sensitizing patients to new allergens and, in extreme cases, eliciting a life-threatening anaphylactic reaction upon direct exposure to the offending allergen(s).
  • In vitro diagnostic testing methods directly measure circulating levels of allergen-specific antibodies in a sample of blood obtained from patients. These methods are generally immunoassay procedures that are reproducible, are equivalent in sensitivity and specificity to well conducted skin prick tests, are unaffected by any of the factors which prevent the use of either of the two in vivo methods, and do not cause anaphylactic events. Immunoassay techniques capable of measuring specific antibody levels to single allergens have been employed for many years (Johansson, S. G. O. and Yman, L., In vitro assay for immunoglobulin E, Coin. Rev. Allergy 6, 93-139, 1988).
  • U.S. Pat. No. 6,087,188 describes a method of detecting an antibody in a sample using paramagnetic particles and a chemiluminescent acridinium compound bound to avidin or streptavidin. The method described in this patent is stated to be useful for the detection of allergens. However, the method is limited to detection of a single allergen in a given sample.
  • Immunoassays In the field of clinical diagnostics there is a broad category of methods available for determining an expanding list of clinically relevant analytes.
  • One such category is immunoassays, which are currently used to determine the presence or concentration of various analytes in biological samples, both conveniently and reliably (The Immunoassay Handbook, edited by David Wild, M Stockton Press, 1994).
  • Immunoassays utilize specific binding agents to target analytes in fluids, where at least one such binding agent is generally labeled with a variety of compounds, including radioisotopes, enzymes and fluorescent or chemiluminescent compounds, that can be measured by radioactive disintegrations, enzymatic induced color- producing substrates, fluorescent output or inhibition and chemiluminescent light output.
  • Such specific binding agents typically include analyte specific antibodies (immunoglobulins) and antibody fragments, receptors, lectins, and genetically or chemically engineered artificial antibodies.
  • Notable immunoassay methods include, for example, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELIZA) (Enzyme-Immunoassay, Edward T. Maggio, CRC Press, 1980), fluorescent immunoassay (FIA) and chemiluminescent assays (CLA) (Luminescent Assays, Perspectives in Endocrinology and Clinical Chemistry, Vol.
  • Immunoassays may be heterogeneous or homogeneous.
  • Heterogeneous immunoassays have been applied to both small and large molecular weight analytes and require separation of bound materials (to be detected or determined) from free materials (which may interfere with that determination).
  • Heterogeneous immunoassays may comprise an antibody or an antigen immobilized on solid surfaces such as plastic microtiter plates, beads, tubes, or the like or on membrane sheets, chips and pieces of glass, nylon, cellulose or the like (Immobilized Enzymes, Antigens. Antibodies, and Peptides, Howard H. Weetall, Marcel Dekker, Inc., 1975).
  • antigen-antibody complexes bound to the solid phase are separated from unreacted and non-specific analyte in solution, generally by centrifugation, filtration, precipitation, magnetic separation or aspiration of fluids from solid phases, followed by repeated washing of the solid phase bound antigen-antibody complex.
  • immunometric “sandwich” assays Immunochemistry of Solid-Phase Immunoassay, John E. Butler, CRC Press, 1991
  • Secondary binding agents are typically immunoglobulin antibodies, antibody fragments, monoclonal antibodies or recombinant antibodies.
  • the analyte is “sandwiched” between the first immobilized antigen or antibody and the labeled secondary binding agent.
  • a subsequent separation and washing is required to remove unbound labeled secondary binding agents.
  • Direct measurement of the labeled, immobilized bound complex or indirect measurement with the use of substrates is then undertaken. It can be appreciated by those familiar with the art of conducting solid phase immunoassays that the procedures are laborious, time consuming and require special equipment or devices for separating immobilized binding agents and analytes.
  • Homogeneous assays are, in general, liquid phase procedures that do not utilize antigens or antibodies that are immobilized on solid materials. Separation and washing steps are not required. The procedures are more commonly involved with the use of fluorescently-labeled antigens or antibodies which upon binding with a target analyte undergo an excitation or quenching of fluorescence emissions, due to the close steric proximity of the binding pair. Homogeneous methods have typically been developed for the detection of haptens, small molecules, such as drugs, hormones and peptides. Macromolecule analytes, such as proteins or peptides with greater than 5000 molecular weight, usually are not determined by homogeneous methods due to a lack of assay sensitivities. A homogeneous method for detection of proteins was reported in U.S. Pat. No. 5,807,675 which required chemical modifications to both binding agents, but is limited to the detection of single analytes.
  • this invention provides a method and a system for simultaneously detecting and quantifying levels of specific immunoglobulin antibodies to a plurality of allergens, using a very small sampling of blood, for the purpose of diagnosing allergy.
  • the invention also provides a method and a system for detecting and quantifying levels of total immunoglobulin E in such a small blood sampling.
  • One method comprises a homogeneous immunoassay for simultaneously detecting and quantifying specific immunoglobulin antibodies to a plurality of allergens in a blood sample, comprising:
  • step (b) thereafter contacting the materials from step (a) with a first conjugate comprising an anti-human IgE or IgG antibody conjugated to a first member of a specific binding pair, said specific binding pair having the capability of amplifying fluorescent signal output, under conditions whereby the anti-human antibody binds to the immunoglobulin antibodies;
  • step (c) thereafter contacting the materials from step (b) with a second conjugate containing a fluorophore moiety coupled to a second member of the specific binding pair under conditions whereby the second member of the specific binding pair binds to the first member of the pair; and
  • step (d) thereafter measuring the fluorescence emitted by the products of step (c).
  • a second method comprises a homogeneous immunoassay for detecting and quantifying total immunoglobulin E levels in a blood sample, comprising:
  • step (b) thereafter contacting the materials from step (a) with a first conjugate comprising an anti-human IgE antibody conjugated to a first member of a specific binding pair, said specific binding pair having the capability of amplifying fluorescent signal output, under conditions whereby the anti-human antibody binds to the anti-human immunoglobulin E;
  • step (c) thereafter contacting the materials from step (b) with a second conjugate containing a fluorophore moiety coupled to a second member of the specific binding pair under conditions whereby the second member of the specific binding pair binds to the first member of the pair; and
  • one system of this invention comprises a system for quantitative determination of specific immunoglobulin (IgE or IgG) antibody levels to allergens in a blood sample comprising:
  • a first conjugate comprising an anti-human IgE or IgG antibody conjugated to a first member of a specific binding pair, said specific binding pair having the capability of amplifying fluorescent signal output, the first conjugate having from 10 to 30 molecules, preferably 15 to 25 molecules, of the first binding pair member conjugated to one molecule of the anti-human antibody;
  • the invention further comprises a system for quantitative determination of total immunoglobulin E levels in a blood sample comprising:
  • a first conjugate comprising an anti-human IgE or IgG antibody conjugated to a first member of a specific binding pair, said specific binding pair having the capability of amplifying fluorescent signal output, the first conjugate having from 10 to 30 molecules, preferably 15 to 25 molecules, of the first binding pair member conjugated to one molecule of the anti-human antibody;
  • the mole ratio of the first conjugate to the second conjugate being from about 1:1 to about 1:5, preferably from about 1:1 to about 1:2.
  • FIG. 1 is a tabulated and graphical view of a specific IgE assay against a panel of ten allergens according to the present invention in accordance with Example 1
  • FIG. 2 is a tabulated and graphical view of a total IgE assay in accordance with Example 2.
  • This invention provides a method and a system for detecting and quantifying specific antibodies to allergens in a blood sample of an individual, for the diagnosis of allergy.
  • in vitro tests for allergy have employed immunoassay methods requiring venous draw samplings of blood to enable testing on a panel of allergens.
  • sample requirements of current methods to determine levels of specific antibodies against a panel of five or more allergens, are from one to three milliliters of blood.
  • the invention limits the sample volume requirements to ten microliters of sample or less, which can be used to test against ten, twenty, forty or more allergens.
  • the amount of sample required is sufficiently small that individuals can remotely obtain a blood sample using a fingerstick or similar procedure.
  • the invention removes the need for obtaining blood samples by venous drawing procedures at physicians' offices, hospital laboratories or clinical reference laboratories.
  • the individual whose blood is to be tested is provided with a kit for collecting and mailing, or otherwise submitting, the blood sample.
  • the kit typically contains one or more devices for puncturing the skin of a finger or other portion of the body (e.g., a fingerprick device) in order to draw some blood, an alcohol or other antiseptic swab for cleaning the area to be punctured, a vial or other small container in which the blood is to be put and sealed, and an appropriate container for mailing the blood sample to the laboratory for testing.
  • the kit also may contain additional items such as adhesives to protect the wound, instructional inserts and labels with individual identification numbers (e.g., PIN) for samples.
  • the exact form and contents of such a kit can vary according to the desires of the laboratory and do not form a part of this invention. Numerous components for such kits are readily available for collection and/or testing of blood (for instance, at home) for various purposes.
  • the providing of a blood sample by the individual without visiting a physician's office or laboratory is not a limiting feature of this invention, but only an advantage of it.
  • the blood sample can well be taken during a visit to a physician's office, and submitted to a laboratory for testing.
  • other advantages of the invention such as the ability to test a small sample, without requiring dilution and/or duplication of tests, are achieved.
  • the invention provides an assay method that combines a solid phase sandwich immunoassay procedure with a homogeneous assay format, where there is no requirement for separation of materials, washing, etc., and no requirement for sample dilution.
  • the assay method quantitates levels of total IgE and specific antibody levels that are essential to allergy diagnosis and to the degree or severity of allergic response to known allergens.
  • a preliminary step in carrying out the method and assembling the system for use in this invention is the preparation and collection of a plurality of solid particles coupled to a plurality of allergens so as to provide a panel of allergens to be tested, for instance, to determine whether an individual's allergic symptoms are mediated by one or more of the allergens under investigation.
  • Each combination of particles with a specific allergen in the panel is distinguishable from combinations of particles with other allergens.
  • a specific allergen can be comprised of extracted proteins, protein fractions purified by chromatographic or affinity chromatographic methods, recombinant proteins or combinations of the above. Allergen mixes, such as two or more grasses, trees or foods, for example, can also be utilized.
  • the capability to distinguish between combinations of particles with different allergens is accomplished by providing a plurality of particles of different types. That is, the particles may be divided into subsets, with each subset being distinguishable from other subsets according to a particular property, characteristic or characteristics.
  • the particles may be divided into subsets where each subset is capable of being distinguished by a specific color or emission spectra, which may be provided by the presence of a fluorochrome or combinations of fluorochromes incorporated within or on it, for example, as described in U.S. Pat. No. 5,981,180.
  • Each subset of the particles is coupled to a specific allergen so that, again, the combination of particles with specific allergen is distinguishable from combinations of particles with other allergens, in accordance with the particular characteristic or characteristics that distinguishes the particle or bead in question from others that are used.
  • the coupling of the allergens to the beads or particles is accomplished by covalent coupling or adsorption methods well known to those familiar with the art and described in the patent and scientific literature (see, for instance, Immunochemistry of Solid-Phase Immunoassay, John E. Butler, CRC Press, 1991 and Immobilized Enzymes, Antigens, Antibodies, and Peptides, edited by Howard H. Weetall, Marcel Dekker, Inc. New York, 1975).
  • the particles themselves are typically spherical (i.e., “beads or microspheres”), with either a rough or a smooth surface, and are prepared as known in the art. They are made various materials, usually non-porous glass, polystyrene, latex or other polymeric materials, and are generally 0.05 micron to 90 micron diameter, preferably 0.5 to 10 micron in diameter, with densities ranging from about 1 to 2 g/mL, preferably close to the density of water.
  • the particle/allergen combinations are preferably stored in a buffered solution containing a protein stabilizer and a bacteriostatic agent, for use as desired.
  • the subsets of individual allergen/particles combinations can be used individually or can be mixed together to form a single assemblage of allergens coupled to particles. This may be done either in advance of conducting assays, so as to provide a preassembled panel of allergens for use in general, or may be done for each individual assay, for instance, in case it is desired to tailor one or more assays to the geographical location or environment of certain patients. Particle concentrations can be determined and adjusted using a conventional counter and the desired number of particles for the assay can be aliquoted.
  • the number of particles in suspension be of limited quantity and in a limited volume to insure assay sensitivity, speed and adaptability to automated microassay formats (if an automated assay is to be used).
  • the particles in suspension should be at a density close to that of water and suspended in a buffered or aqueous medium sufficient in volume to allow molecular motion and molecular contact and to be adaptable to microassay formats.
  • the panel for testing can contain, overall, as few as about 5, and up to about 100, specific allergens or mixtures of allergens; however, panels of 10 to 40 allergens are more common. It is preferred that for each test, the sample is added to a suspension containing from about 1000 particles to about 4000 particles of each set of particle-coupled allergen or mixture of allergens, preferably in a small volume, for example, a volume of from about 1 to about 50 ⁇ L, preferably from about 5-25 ⁇ L.
  • a test with a panel of 10 allergens would contain about 10,000 particles, in 5-20 microliters of a buffered solution.
  • a test with a panel of 20 allergens would incorporate about 20,000 particles in a 5-20 microliter volume. Increasing the number of particles in an assay decreases the amount of time required to identify and read the particles.
  • the particles thus prepared are contacted with an individual's blood sample.
  • the blood sample will have a volume of from about 1 to about 25 ⁇ L, preferably from about 1 to about 5 ⁇ L.
  • incubation conditions such as time, temperature, pH, ionic strength of reagents, and the like, and the conditions of a given assay will be chosen as known in the art to optimize the sensitivity and specificity of the test and generally suit the ease of use of the protocol and its adaptability to automation.
  • such conditions include a temperature of from about 18 to about 37° C., preferably ambient temperatures of from about 18 to about 25° C., and a time of from about 15 minutes to about 24 hours, preferably 1 hour or less.
  • anti-human antibody coupled to particles are contacted with an individual's sample.
  • the sample will have a volume of from about 1 to about 5 ⁇ L, preferably from about 1 to about 2 ⁇ L. Incubation times and conditions are determined as described herein.
  • a first conjugate comprising an antibody to the specific analyte, such as anti-human IgE, that is coupled to the first member of a specific binding pair is sequentially added to the mixture. Separation of the solid phase particles from the reaction medium and sample is not required.
  • the specific binding pair is a pair of molecules that have binding specificity for one another. Examples of types of specific binding pairs are biotin-avidin, biotin-streptavidin, digoxin-antidigoxin, and complementary homopolynucleotides poly (dA)-poly (dT) (described in U.S. Pat. No. 6,245,513).
  • the biotin-streptavidin specific binding pair is a preferred embodiment of this invention.
  • a plurality of one member of the specific binding pair is coupled (conjugated) to the antibody (e.g., anti-human IgE).
  • the second member of the specific binding pair e.g., streptavidin
  • the binding of the second specific binding pair member, streptavidin, to the plurality of biotins conjugated to antibody amplifies the fluorescent signal, providing greater assay sensitivity.
  • Biotinylation of antibody can be carried out by techniques well known in the art.
  • antibody-biotin conjugates either obtained commercially or prepared contain 5 to 10 biotin molecules per antibody molecule. In this preferred application, optimum assay sensitivity is obtained when from about 10 to about 30 biotin molecules are coupled per antibody molecule, preferably, about 15-25 biotin molecules per antibody molecule.
  • binding of analytes to antigens or antibodies is influenced by incubation conditions such as time, temperature, pH, ionic strength of reagents, and the like, and the conditions of a given assay will be chosen as known in the art to optimize the sensitivity and specificity of the test and generally suit the ease of use of the protocol and its adaptability to automation.
  • incubation conditions such as time, temperature, pH, ionic strength of reagents, and the like
  • such conditions of a given assay will be chosen as known in the art to optimize the sensitivity and specificity of the test and generally suit the ease of use of the protocol and its adaptability to automation.
  • such conditions include a temperature of from about 18 to about 37° C., preferably ambient temperatures of from about 18 to about 25° C., and a time of from about 15 minutes to about 24 hours, preferably 1 hour or less.
  • secondary binding agents will bind to the analyte-particle complex through normal antigen-antibody binding forces.
  • a second conjugate containing the second member of the specific binding pair conjugated to a fluorophore, is added to the mixture.
  • the second binding pair member is preferably avidin or streptavidin, most preferably streptavidin, though, as mentioned above, it may be anti-digoxin or materials described in U.S. Pat. No. 6,245,513.
  • Covalent attachment of fluorescent labels to avidin or streptavidin may be effected by a variety of techniques previously described in patent and scientific literature (Haugland, R. P., Bhalagat, M. K., Preparation of avidin conjugates, Methods Mol. Biol. 1998; 80:185-96).
  • Typical fluorescent moieties are described in Chapter 3 of the Manual of Clinical Laboratory Immunology, supra.
  • the conjugates may be obtained from a commercial sources. Fluorescent dyes such as fluorescein, the arylsulfonate cyanine dyes, phycobiliprotein dyes, bodipy dyes and the like, may be used. If the particle subsets are distinguished from one another on the basis of incorporation of fluorochromes, the dyes used in the conjugates are selected so as to have fluorescent emissions that are distinct from, and do not interfere with, the emission spectra of the particle subsets.
  • a preferred type of fluorescent material is a class of compounds known as phycobiliproteins, more particularly the phycoerytherins, the phycocyanins, and the allophycocyanins, most preferably the phycoerytherins.
  • Phycoerytherin conjugates have sensitivities ranging from five to ten times greater than that of corresponding fluorescein conjugates, with quantum yields of up to 0.98 and extinction coefficients of up to 2.4 million (cm ⁇ 1 M ⁇ 1 ). Most preferred of these is R-phycoerytherin.
  • the molecular ratio of fluorescent dye to second binding pair member generally can range from about 1:1 to up to about greater than 5:10 with molecular weights ranging from about 300,000 to 3,000,000 or more daltons.
  • the preferred molecular weight of the second conjugate generally is about 400,000 to about 1,000,000 daltons.
  • the materials are incubated under appropriate conditions for binding of the binding pair members. These, as are known in the art, typically include a temperature of from about 18° C. to about 45° C., preferably from about 18° C. to about 25° C. and a time of from about 15 minutes to about 24 hours, preferably from about 15 minutes to about 3 hours.
  • the fluorescently labeled antibodies bind to the particles through the first and second binding partners, and through the binding of antibody to analyte antigen. They thus can be detected and measured by application of excitation energy having a wavelength selected to excite the chosen fluorescent label, where the emission spectra that is generated is distinct from the emission spectra incorporated in the particles.
  • An important feature of the overall method of this invention is that the immunoassay can be conducted without sample dilution. Furthermore, by limiting the sample size to 1-25 ⁇ L, preferably 1-5 ⁇ L, a homogeneous assay format with solid phase particles, with appropriate assay range and sensitivity, is now possible.
  • the individual who submitted the sample, or another individual acting with his or her permission can obtain access to the test results over the Internet, or a similar global computer system, for instance, from a Web site via a Web server.
  • Web server may also refer to a plurality of servers organized to handle a large number of requests for a Web server, i.e., a distributed Web server system.
  • Web site is often used to refer to a collection of Web servers organized by a business entity or other entity for their purposes. A user is said to “go to” or “access” a Web site when the user directs his or her Web client to make a request of one or the site's Web servers and display the response to the user (even though the user and the Web client do not actually move physically).
  • Web site often refers to the Web server or servers that respond to requests from Web clients, even though “site” does not necessarily refer to the physical location of the Web servers. In fact, in many cases, the servers that serve up a Web site might be distributed physically to avoid downtime when local outages of power or network service occur.
  • Web site typically refers to a collection of pages maintained by a common maintainer for presentation to visitors, whether the collection is maintained on one physical server at one physical location or is distributed over many locations and/or servers.
  • the pages (or the data/program code needed to generate the pages dynamically) need not be created by the common maintainer of the collection of pages.
  • Such a maintainer of the collection of pages is typically referred to as the Web site operator.
  • Web site also includes Web sites connected to the Web clients via an intranet, wireless access protocol (WAP) network, a local area network (LAN), a wide area network (WAN), a virtual private network (VPN) or another network arrangement.
  • WWW wireless access protocol
  • LAN local area network
  • WAN wide area network
  • VPN virtual private network
  • Web typically refers to “World Wide Web” (or just “the WWW”), a name given to the collection of hyperlinked documents accessible over the Internet using HTTP.
  • WWW World Wide Web
  • “Web” might refer to the World Wide Web, a subset of the World Wide Web, a local collection of hyperlinked pages, or the like.
  • the submitter of the sample can obtain access to information respecting the test results, on a Web site, Web “page” or the like.
  • a Web page typically consists of certain information.
  • Web pages include both static pages and dynamic pages.
  • Static pages are pages that are stored on the server, or in storage accessible by the server, prior to the request and are sent from storage to the client in response to a request for that page.
  • Dynamic pages are pages that are generated, in whole or in part, upon receipt of a request. For example, where the page is a view of data from a database, a server might generate the page dynamically using rules or templates and data from the database where the particular data used depends on the particular request made.
  • the submitter can be assigned a password or code (PIN) that permits access to his or her test results on a web page or within a database that is maintained by, or for, the testing organization (e.g. testing laboratory).
  • PIN password or code
  • a submitter may also receive the test results without having to visit a physician's office, and may then provide such information to a physician of his or her choice, at his or her convenience.
  • Computer software that may be used to maintain, and provide access to, such test results, is available from vendors, and does not form a part of this invention.
  • Extracted allergens typically from trees, grasses, molds, foods, danders and the like, are coupled to microparticles by covalent or adsorbed procedures. Each allergens extract is coupled to a unique subset of particles, with each subset distinguishable by an incorporated fluorescent emissions or particle size where such differing characteristics can be recognized by appropriate instrumentation.
  • the allergen-coupled particle subsets are stored individually or the subsets are combined as a panel of allergens in a buffered medium with protein stabilizers and bacteriostatic agents.
  • each allergen particle subset represented by 1,000 particles
  • 10 ⁇ L of the combined particle subsets totaling 10,000 particles, each allergen particle subset represented by 1,000 particles, are placed in a microfuge tube or microtiter well.
  • anti-human IgE is coupled to microparticles by covalent or adsorbed procedures, and 1,000 particles are placed in a microfuge tube or microtiter well for each test.
  • Blood, plasma or serum collected from an individual by means of the fingerstick collection kit as described herein, or other collection methods is appropriately labeled or bar coded for identification.
  • a few drops of collected blood, plasma or serum is adequate for determining specific antibody levels to a panel of allergens.
  • a 5 ⁇ L sample is added to the allergen-coupled particles residing in the microfuge tubes or microtiter wells.
  • the combined mixture can be briefly vortexed (with microfuge tubes) or shaken (microtiter plate shaker) and incubated for 1 hour at room temperature.
  • a 1 ⁇ L sample is added to the anti-human IgE coupled particles residing in the microfuge tube or microtiter well for each test.
  • the resulting mixture of materials is subjected to a reading via appropriate flow cytometric instrumentation, such as the Luminex Corporation Luminex 100 System® or the Becton-Dickinson Immunosytometry FACSCAN® instruments, which simultaneously determines the particle subset by the particle incorporated fluorescent emissions or particle size and measures the emission spectra of the R-phycoerytherin attached to the particle via antigen-antibody and biotin-streptavidin binding as previously described.
  • appropriate flow cytometric instrumentation such as the Luminex Corporation Luminex 100 System® or the Becton-Dickinson Immunosytometry FACSCAN® instruments, which simultaneously determines the particle subset by the particle incorporated fluorescent emissions or particle size and measures the emission spectra of the R-phycoerytherin attached to the particle via antigen-antibody and biotin-streptavidin binding as previously described.
  • the biotinylated antibody solution is purified on a Sephadex G-25 (Pharmacia, Sweden) desalting column equilibrated with Phosphate Buffered Saline (PBS), pH 7.2, containing 0.1% Sodium Azide (Sigma)
  • Carboxylated polystyrene microspheres (Luminex Corporation, TX, USA) are suspended in 80 L of 0.1M MES Buffer (Fisher Scientific, PA, USA) pH 6.1. To the suspension is added 10 ⁇ L of N-Hydroxysulfosuccinimide sodium salt (Pierce Chemicals, IL, USA) 50 mg/mL and 10 ⁇ L of 1-(3-dimethylaminopropyl)-3-Ethyl-carbodiimide hydrochloride (Pierce Chemicals, IL, USA) 5 mg/mL. The solution is vortexed and let stand in the dark for 15-20 minutes at ambient temperature.
  • the activated microspheres are centrifuged for 5 minutes at 5000 g and the supernatant is aspirated.
  • the microspheres are then resuspended in 250 ⁇ L of 0.1M PBS solution (0.1 M Sodium Phosphate, 0.14 M NaCl) pH 7.3. The mixture is vortexed, the activated microspheres centrifuged and the supernatant aspirated as before.
  • 0.1M PBS solution 0.1 M Sodium Phosphate, 0.14 M NaCl
  • the activated microspheres are then resuspended in 250 ⁇ L of allergen extract, purified proteins or recombinant proteins. Then the mixture is rotated (protected from light) for at least 1 hour at ambient temperature and centrifuged for 5 minutes at 5000 g. The supernatant is aspirated and the microspheres resuspended in 250 ⁇ L of PBS/HSA Buffer pH 7.3, containing 0.01 M PBS and 0.02 mg/mL Human Serum Albumin (Sigma, Mo., USA), and vortexed to wash the microspheres of residual allergen extract. The supernatant is again centrifuged and aspirated as previously described.
  • microsphere concentrations in solution is determined by counting using a hemocytometer (Fisher Scientific, PA, USA) and microscope (Nippon Kogaku, Japan).
  • Subsets of fluorescently labeled microspheres obtained from Luminex Corporation were covalently coupled with Alternaria mold, Bermuda grass, Timothy grass, Cat dander, Mountain Cedar tree, Egg white, Milk, Wheat, Ragweed and Mite extracted proteins. Serum samples obtained from atopic and non-atopic individuals were assayed against this panel of ten allergens. A 5 ⁇ L sample was added to 10 ⁇ L of suspended allergen-coupled microspheres placed in V-bottom polypropylene microtiter wells (Evergreen Scientific, CA, USA) or 0.5 mL polypropylene microfuge tubes (Evergreen Scientific, CA, USA).
  • FIG. 1 shows the results from two non-allergic and three allergic individuals with positive specific IgE responses to a panel of ten allergens.
  • a 5 ⁇ L volume of sample from each individual was used in a three-hour homogeneous assay procedure to simultaneously measure specific IgE levels to the ten allergens coupled to microspheres.
  • Measurement of fluorescent intensity signals using the Luminex 100 System was used to report the results in either IU/mL or class units as interpolated from a six-point standard curve from 0.35 IU/mL.
  • Response interpretations range from Negative to Extremely High based on class levels obtained.
  • Goat anti-human IgE (Bethyl Laboratories, TX, USA) was coupled to microsphere particles (Luminex Corporation, TX, USA) following the same procedure described above for coupling allergens. Serum samples were obtained from individuals obtained by the methods described above. A 1.0 ⁇ L volume of standard (secondary standards calibrated against the WHO IgE Standard 75/502) or individual sample was added to 25 ⁇ L of suspended microspheres coupled with antibody in microtiter wells, briefly shaken as described above and incubated for 1 hour. A 40 ⁇ L volume of anti-human IgE -biotin conjugate was subsequently added to the contents of each well and incubated for 1 hour.
  • FIG. 2 shows the results of the Total IgE Standard Curve and Total IgE results of ten individuals.
  • a 1 ⁇ L volume of sample from each individual was used in a three-hour homogeneous assay procedure.
  • the amount of total IgE bound to anti-human IgE-coupled microspheres was determined by measuring the signal intensity from the fluorescent reporter signals using the Luminex 100 System and interpolated from a six-point standard curve from 2-2000 IU/mL. Expected results on samples were provided by a reference laboratory using an alternative essay procedure.

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US10/006,483 US20030109067A1 (en) 2001-12-06 2001-12-06 Homogeneous immunoassays for multiple allergens
DK02786929.6T DK1461618T3 (da) 2001-12-06 2002-12-06 Homogene immununoassays til flere allergener
PCT/US2002/039111 WO2003050539A1 (en) 2001-12-06 2002-12-06 Homogeneous immunoassays for multiple allergens
ES02786929T ES2340667T3 (es) 2001-12-06 2002-12-06 Inmunodeficiencia homogeneos para multiples alergenos.
AU2002351281A AU2002351281A1 (en) 2001-12-06 2002-12-06 Homogeneous immunoassays for multiple allergens
EP02786929A EP1461618B1 (en) 2001-12-06 2002-12-06 Homogeneous immunoassays for multiple allergens
AT02786929T ATE457459T1 (de) 2001-12-06 2002-12-06 Homogene immunoassays für mehrere allergene
DE60235324T DE60235324D1 (de) 2001-12-06 2002-12-06 Homogene immunoassays für mehrere allergene
JP2003551541A JP2005512091A (ja) 2001-12-06 2002-12-06 多重アレルゲンに対する均質免疫分析
US10/387,790 US20030232397A1 (en) 2001-12-06 2003-03-12 Homogeneous immunoassays for multiple allergens
US11/786,193 US7491553B2 (en) 2001-12-06 2007-04-10 Homogeneous immunoassays for multiple allergens
US12/316,838 US7892853B2 (en) 2001-12-06 2008-12-16 Homogeneous immunoassays for multiple allergens
US13/006,394 US8034632B2 (en) 2001-12-06 2011-01-13 Homogeneous immunoassays for multiple allergens
US13/006,392 US8034631B2 (en) 2001-12-06 2011-01-13 Systems for homogeneous immunoassays for multiple allergens
US13/006,398 US8093066B2 (en) 2001-12-06 2011-01-13 Binding reagent system for homogeneous immunoassays for multiple allergens
US13/312,977 US8349620B2 (en) 2001-12-06 2011-12-06 Highly sensitive immunoassay systems for detecting multiple allergens
US13/335,808 US20120264230A1 (en) 2001-12-06 2011-12-22 home or point-of-care allergy testing

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US11/786,193 Expired - Fee Related US7491553B2 (en) 2001-12-06 2007-04-10 Homogeneous immunoassays for multiple allergens
US12/316,838 Expired - Fee Related US7892853B2 (en) 2001-12-06 2008-12-16 Homogeneous immunoassays for multiple allergens
US13/006,398 Expired - Fee Related US8093066B2 (en) 2001-12-06 2011-01-13 Binding reagent system for homogeneous immunoassays for multiple allergens
US13/006,392 Expired - Fee Related US8034631B2 (en) 2001-12-06 2011-01-13 Systems for homogeneous immunoassays for multiple allergens
US13/006,394 Expired - Fee Related US8034632B2 (en) 2001-12-06 2011-01-13 Homogeneous immunoassays for multiple allergens
US13/312,977 Expired - Fee Related US8349620B2 (en) 2001-12-06 2011-12-06 Highly sensitive immunoassay systems for detecting multiple allergens
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US12/316,838 Expired - Fee Related US7892853B2 (en) 2001-12-06 2008-12-16 Homogeneous immunoassays for multiple allergens
US13/006,398 Expired - Fee Related US8093066B2 (en) 2001-12-06 2011-01-13 Binding reagent system for homogeneous immunoassays for multiple allergens
US13/006,392 Expired - Fee Related US8034631B2 (en) 2001-12-06 2011-01-13 Systems for homogeneous immunoassays for multiple allergens
US13/006,394 Expired - Fee Related US8034632B2 (en) 2001-12-06 2011-01-13 Homogeneous immunoassays for multiple allergens
US13/312,977 Expired - Fee Related US8349620B2 (en) 2001-12-06 2011-12-06 Highly sensitive immunoassay systems for detecting multiple allergens
US13/335,808 Abandoned US20120264230A1 (en) 2001-12-06 2011-12-22 home or point-of-care allergy testing

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