Binding reagent system for homogeneous immunoassays for multiple allergens
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.
1. A binding agent system for use in a homogeneous immunoassay testing for an allergic response in a patient, comprising:
labeled subsets of specific allergen-coupled particles for combining with an undiluted serum sample from the patient under conditions that enable binding of the allergen-coupled particles to allergen-specific IgE antibodies from the patient's serum sample, the combining creating a response mixture;
a first binding agent for adding to the response mixture, the first binding agent comprising an anti-human IgE antibody covalently bound to a first member of a binding pair; and,
a labeled second binding agent for adding to the response mixture containing the first binding agent, the labeled second binding agent comprising a second member of the binding pair and a label, wherein the second member of the binding pair has an affinity for binding to the first member of the binding pair;
wherein,
the first binding agent and the second binding agent are preselected to enable detection of anti-human IgE antibodies at a detection sensitivity ranging from about 10 −9 g/ml to about 10 −12 g/ml, and the undiluted serum is limited to having a volume of about 0.5 μL to about 25 μL;
the molecular ratio of anti-human antibody to the first member of the binding pair in the first binding agent is from about 1:10 to about 1:30, the second binding agent has a molecular weight of between about 400,000 and about 1,000,000 Daltons, and the selection of the first binding agent, the second binding agent, and serum volume has the function of avoiding (i) quenching of a signal from the label of the labeled second binding agent and (ii) high dose hook effects;
the detection sensitivity ranging from 10 −9 g/ml to 10 −12 g/ml is measured under homogeneous assay conditions; and,
the amounts of the allergen-specific antibodies from the serum sample that are bound to specific allergens are determined by (i) identifying and distinguishing the labeled particles to identify the subsets of specific allergens and (ii) measuring the amount of labeled second binding agent in each of the subsets of allergens.
2. The system of claim 1 , wherein the labeled particles are fluorescently labeled microspheres, and the undiluted serum sample ranges in volume from about 1.0 μL to about 10 μL.
3. The system of claim 1 , wherein the first binding agent comprises an anti-human IgE-antibody-biotin conjugate having about 15-25 biotin molecules per antibody molecule, the second binding agent comprises a streptavidin-phycoerytherin conjugate, and the mole ratio of the first binding agent to the second binding agent ranges from about 1:1 to about 1:5.
4. The system of claim 1 , wherein the labeled particles enable identification of subsets of allergens, and the subsets compose a panel of allergens having from about 2 specific allergens to about 100 specific allergens.
5. The system of claim 1 , wherein the system further comprises a kit having a device for puncturing the skin and obtaining a blood sample, and a container for holding and transmitting the blood sample to a laboratory for extracting the serum sample and measuring the allergic response of the patient to an allergen or panel of allergens.
6. The system of claim 1 , wherein the system further comprises a computer network component having testing results for the patient on a non-transitory computer readable storage medium accessible by the patient to obtain testing results.
7. The system of claim 1 , wherein the first binding agent comprises an anti-human antibody-digoxin conjugate.
8. A binding agent system for use in a homogeneous immunoassay testing for an allergic response in a patient, comprising:
labeled subsets of specific allergen-coupled particles for combining with an undiluted serum sample from the patient under conditions that enable binding of the allergen-coupled particles to allergen-specific antibodies from the patient's serum sample, the combining creating a response mixture;
a first binding agent for adding to the response mixture, the first binding agent comprising an anti-human antibody covalently bound to a first member of a binding pair; and,
a labeled second binding agent for adding to the response mixture containing the first binding agent, the labeled second binding agent comprising a second member of the binding pair and a label, wherein the second member of the binding pair has an affinity for binding to the first member of the binding pair;
wherein,
the first binding agent and the second binding agent are preselected to enable detection of anti-human antibodies at a detection sensitivity ranging from about 10 −9 g/ml to about 10 −12 g/ml, and the undiluted serum is limited to having a volume of about 0.5 μL to about 25 μL;
the molecular ratio of anti-human antibody to the first member of the binding pair in the first binding agent is from about 1:10 to about 1:30, the second binding agent has a molecular weight of between about 400,000 and about 1,000,000 Daltons, and the selection of the first binding agent, the second binding agent, and serum volume has the function of avoiding (i) quenching of a signal from the label of the labeled second binding agent and (ii) high dose hook effects; and
the detection sensitivity ranging from 10 −9 g/ml to 10 −12 g/ml is measured under homogeneous assay conditions; and,
the amounts of the allergen-specific antibodies from the serum sample that are bound to specific allergens are determined by (i) identifying and distinguishing the labeled particles to identify the subsets of specific allergens and (ii) measuring the amount of labeled second binding agent in each of the subsets of allergens.
9. The system of claim 8 , wherein the labeled particles are fluorescently labeled microspheres, and the undiluted serum sample ranges in volume from about 1.0 μL to about 10 μL.
10. The system of claim 8 , wherein the first binding agent comprises an anti-human antibody-biotin conjugate having about 15-25 biotin molecules per antibody molecule, the second binding agent comprises a streptavidin-phycoerytherin conjugate, and the mole ratio of the first binding agent to the second binding agent ranges from about 1:1 to about 1:5.
11. The system of claim 8 , wherein the labeled particles enable identification of subsets of allergens, and the subsets compose a panel of allergens having from about 2 specific allergens to about 100 specific allergens.
12. The system of claim 8 , wherein the system further comprises a kit having a device for puncturing the skin and obtaining a blood sample, and a container for holding and transmitting the blood sample to a laboratory for extracting the serum sample and measuring the allergic response of the patient to an allergen or panel of allergens.
13. The system of claim 8 , wherein the system further comprises a computer network component having testing results for the patient on a non-transitory computer readable storage medium accessible by the patient to obtain testing results.
14. The system of claim 8 , wherein the first binding agent comprises an anti-human antibody-digoxin conjugate.
15. The system of claim 8 , wherein the allergen-specific antibodies from the patient's serum sample are allergen-specific IgG antibodies, and the anti-human antibody in the first binding agent is an anti-human IgG antibody.
16. A binding agent system for use in a homogeneous immunoassay testing for an allergic response in a patient, comprising:
labeled subsets of specific allergen-coupled particles for combining with an undiluted serum sample from the patient under conditions that enable binding of the allergen-coupled particles to allergen-specific IgG antibodies from the patient's serum sample, the combining creating a response mixture;
a first binding agent for adding to the response mixture, the first binding agent comprising an anti-human IgG antibody covalently bound to a first member of a binding pair; and,
a labeled second binding agent for adding to the response mixture containing the first binding agent, the labeled second binding agent comprising a second member of the binding pair and a label, wherein the second member of the binding pair has an affinity for binding to the first member of the binding pair;
wherein,
the first binding agent and the second binding agent are preselected to enable detection of anti-human IgG antibodies at a detection sensitivity ranging from about 10 −9 g/ml to about 10 −12 g/ml, and the undiluted serum is limited to having a volume of about 0.5 μL to about 25 μL;
the molecular ratio of anti-human antibody to the first member of the binding pair in the first binding agent is from about 1:10 to about 1:30, the second binding agent has a molecular weight of between about 400,000 and about 1,000,000 Daltons, and the selection of the first binding agent, the second binding agent, and serum volume has the function of avoiding (i) quenching of a signal from the label of the labeled second binding agent and (ii) high dose hook effects;
the detection sensitivity ranging from 10 −9 g/ml to 10 −12 g/ml is measured under homogeneous assay conditions; and,
the amounts of the allergen-specific antibodies from the serum sample that are bound to specific allergens are determined by (i) identifying and distinguishing the labeled particles to identify the subsets of specific allergens and (ii) measuring the amount of labeled second binding agent in each of the subsets of allergens.
17. The system of claim 16 , wherein the labeled particles are fluorescently labeled microspheres, and the undiluted serum sample ranges in volume from about 1.0 μL to about 10 μL.
18. The system of claim 16 , wherein the first binding agent comprises an anti-human IgG-antibody-biotin conjugate having about 15-25 biotin molecules per antibody molecule, the second binding agent comprises a streptavidin-phycoerytherin conjugate, and the mole ratio of the first binding agent to the second binding agent ranges from about 1:1 to about 1:5.
19. The system of claim 16 , wherein the labeled particles enable identification of subsets of allergens, and the subsets compose a panel of allergens having from about 2 specific allergens to about 100 specific allergens.
20. The system of claim 16 , wherein the system further comprises a kit having a device for puncturing the skin and obtaining a blood sample, and a container for holding and transmitting the blood sample to a laboratory for extracting the serum sample and measuring the allergic response of the patient to an allergen or panel of allergens.
21. The system of claim 16 , wherein the system further comprises a computer network component having testing results for the patient on a non-transitory computer readable storage medium accessible by the patient to obtain testing results.
22. The system of claim 16 , wherein the first binding agent comprises an anti-human IgG-antibody-digoxin conjugate.