Homogeneous immunoassays for multiple allergens
First Claim
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1. A method for simultaneously measuring specific immunoglobin antibody levels to from 2 to about 100 allergens in a blood sample, said method comprising:
- (a) collecting a blood sample of from about 10 to about 100 μ
L from an individual;
(b) performing a homogeneous immunoassay on the blood sample or a portion thereof, wherein the immunoassay is capable of determining specific immunoglobin antibody levels to from 2 to about 100 allergens; and
(c) determining the said specific immunoglobin antibody levels.
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Abstract
A homogeneous immunoassay method and system for quantitative determination of total immunoglobulin E and specific antibody levels to a plurality of allergens, in which a relatively small sampling of blood is required. The method utilizes relatively small microparticles in aqueous suspension. The immunoassay procedure is an immunometric sandwich procedure preferably utilizing biotin-streptavidin signal amplification techniques and R-phycoerytherin fluorescent labels.
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Citations
86 Claims
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1. A method for simultaneously measuring specific immunoglobin antibody levels to from 2 to about 100 allergens in a blood sample, said method comprising:
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(a) collecting a blood sample of from about 10 to about 100 μ
L from an individual;
(b) performing a homogeneous immunoassay on the blood sample or a portion thereof, wherein the immunoassay is capable of determining specific immunoglobin antibody levels to from 2 to about 100 allergens; and
(c) determining the said specific immunoglobin antibody levels.
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2. A system for simultaneously measuring a plurality of specific immunoglobin antibody levels by use of a homogeneous immunoassay, said system comprising:
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(a) a plurality of particles coupled to from 2 to about 100 allergens;
(b) a first conjugate comprising an anti-human IgE or IgG antibody conjugated to a first member of a specific binding pair; and
(c) a second conjugate comprising a second specific binding pair member that binds to the first specific binding pair member;
the system being capable of conducting simultaneous quantitative determination of specific immunoglobin antibody levels to from 2 to about 100 allergens, by a homogeneous immunoassay.
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3. A system for quantitative determination of specific immunoglobulin antibody levels to allergens in a blood sample comprising:
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(a) a plurality of particles coupled to a plurality of allergens, the particles being in suspension in from about 1 to about 50 μ
L of an aqueous medium, each combination of particles with a specific allergen being distinguishable from combinations of the particles with other allergens;
(b) 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 of the first binding pair member conjugated to one molecule of the anti-human antibody;
(c) a second conjugate comprising a second specific binding pair member that binds to the first specific binding pair member, the second member being coupled to a fluorophore moiety;
(d) the mole ratio of first conjugate to the second conjugate being from about 1;
1 to about 1;
5. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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28. A homogeneous immunoassay method for simultaneously detecting and quantifying specific immunoglobulin antibody levels to a plurality of allergens in a blood sample, comprising
(a) contacting a blood sample having a volume of from about 1 to about 25 μ - L with a plurality of particles coupled to a plurality of allergens, the particles being in suspension in from about 1 to about 50 μ
L of an aqueous medium, each combination of particles with specific allergen being distinguishable from combinations of the particles with other allergens, under conditions whereby immunoglobulin antibodies present in the blood sample that bind specifically to one or more of the allergens are bound to the particles;
(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;
(c) thereafter contacting the materials from step (b) with a second conjugate containing a fluorophore moiety bound to a second specific binding pair member that binds to the first specific binding pair member; and
(d) thereafter determining levels of specific immunoglobulin antibody in the materials from step (c) by simultaneously determining the fluorescent emission signals of the particle subsets and measuring the fluorescent emission signals of the fluorophore moiety. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
- L with a plurality of particles coupled to a plurality of allergens, the particles being in suspension in from about 1 to about 50 μ
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49. A system for quantitative determination of total immunoglobulin E levels in a blood sample comprising:
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(a) A plurality of particles coupled to anti-human IgE antibody, the particles being in suspension in from about 1 to about 50 μ
L of an aqueous medium;
(b) a first conjugate comprising an anti-human antibody and a first member of a specific binding pair, said specific binding pair having the capability of amplifying fluorescent signal output;
(c) a second conjugate comprising a phycobiliprotein and a second specific binding pair member that binds specifically to the first specific binding pair member and amplifies the fluorescent signal output;
(d) the mole ratio of the first conjugate to the second conjugate being from about 1;
1 to about 1;
5. - View Dependent Claims (50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68)
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69. A homogeneous immunoassay method for detecting and quantifying total immunoglobulin E antibodies in a blood sample, comprising
(a) contacting a blood sample having a volume of from about 0.5 to about 10 μ - L with a plurality of particles coupled to anti-human immunoglobulin E, the particles being in suspension in from about 1 to about 50 μ
L of an aqueous medium.(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;
(c) thereafter contacting the materials from step (b) with a second conjugate containing a fluorophore moiety bound to a second specific binding pair member that binds to the first specific binding pair member; and
(d) thereafter determining levels of specific immunoglobulin antibody in the materials from step (c) by simultaneously determining the fluorescent emission signals of the particles and measuring the fluorescent emission signals of the fluorophore moiety bound to the particles. - View Dependent Claims (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85)
- L with a plurality of particles coupled to anti-human immunoglobulin E, the particles being in suspension in from about 1 to about 50 μ
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86. A method for conducting and providing tests for the presence of specific antibodies to allergens or for total IgE in a sample, comprising:
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(a) providing a blood collection kit so as to enable an individual to provide a sample of blood for use in the method by pricking the skin to draw blood, collecting a sample of said blood and submitting said sample for use in the method;
(b) conducting a homogeneous immunoassay to detect the presence of antibodies to specific allergens or of total IgE in the sample from step (a);
(c) entering results of the immunoassay from step (b) into a database; and
(d) providing the individual access to the results of the immunoassay in the database of step (c) via a Web site or Web page of a global computer system.
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Specification