RAPID MAGNETIC FLOW ASSAYS
2 Assignments
0 Petitions
Accused Products
Abstract
Disclosed is an improvement in methods for nucleic acid and immunological bioassays. The methods comprise a step for “sweeping” paramagnetic bead: target molecule complexes so as to capture them with an affinity capture agent on a test pad by moving a magnetic force field from outside to inside the test pad area so as to bring into contact the paramagnetic complexes with the capture agent, while sweeping any unbound paramagnetic material off the test pad by moving the magnetic field from inside to outside the test pad area. Surprisingly, the paramagnetic complexes are rapidly affinity-extracted from the moving magnetic field.
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Citations
56 Claims
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1-4. -4. (canceled)
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5. A method for multiplex target nucleic acid detection by heterogeneous binding assay, comprising:
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a) preparing an amplification reagent comprising an amplification primer set having a first primer tagged with a peptidyl hapten tag and a second primer tagged with a ligand tag, wherein said first primer and said second primer are complementary to flanking sequences of a nucleic acid target sequence to be assayed; b) preparing a paramagnetic bead reagent comprising a paramagnetic microbead with a binding agent, wherein said binding agent has a binding affinity for said ligand tag of said second primer; c) preparing a test pad area with immobilized capture antibody, wherein said capture antibody has a binding affinity for said peptidyl hapten tag of said first primer; d) processing a biological sample to release a nucleic acid fraction; e) contacting said nucleic acid fraction with said amplification reagent; f) performing an amplification to yield amplification products; and g) assaying said amplification products for a two-tailed amplicon labeled with said peptidyl hapten tag of said first primer on a first end of said two-tailed amplicon and said ligand tag of said second primer on a second end of said two-tailed amplicon by; i) contacting said amplification products with said paramagnetic bead reagent, thereby binding said ligand tag of said second end of said two-tailed amplicon to said binding agent of said paramagnetic bead reagent to yield a two-tailed amplicon paramagnetic bead complex; ii) sweeping said two-tailed amplicon paramagnetic bead complex into close contact with said test pad area by moving a magnetic force field from outside to inside said test pad area, thereby binding said peptidyl hapten tag of said first end of said two-tailed amplicon of said two-tailed amplicon paramagnetic bead complex to said capture antibody of said test pad area to yield an immunoimmobilized paramagnetic reporter complex; iii) sweeping from said test pad area any unbound two-tailed amplicon paramagnetic bead complex by moving said magnetic force field from inside to outside said test pad area; and iv) detecting the presence of said immunoimmobilized paramagnetic reporter complex on said test pad area. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 49, 50)
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14-33. -33. (canceled)
- 34. A molecular detection complex comprising a two-tailed amplicon having a first end and a second end, said first end comprising a first primer covalently conjugated with a peptidyl hapten tag, and said second end comprising a second primer covalently conjugated with a ligand tag, wherein said first end further comprises a peptidyl hapten-bound anti-peptidyl hapten antibody immobilized on a solid phase, and wherein said second end further comprises a ligand-bound ligand binding agent-coated reporter group.
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37-43. -43. (canceled)
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44. A method for rapid bioassay comprising:
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a) preparing an amplification primer set comprising a first primer comprising a peptidyl hapten tag and a second primer comprising a ligand tag; b) forming a two-tailed amplicon product comprising said peptidyl hapten tag of said first primer on a first end and said ligand tag of said second primer on a second end by amplifying a nucleic acid target with said amplification primer set; c) complexing said two-tailed amplicon product to a reporter group having a binding affinity for said ligand tag to yield a two-tailed amplicon reporter group complex; d) capturing said two-tailed amplicon reporter group complex on a solid phase having an immobilized capture antibody, wherein said capture antibody has a binding affinity for said peptidyl hapten tag, to yield an immobilized reporter group complex; and
,e) detecting said immobilized reporter group complex. - View Dependent Claims (45, 46, 47, 48)
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51. A method for multiplex target nucleic acid detection by heterogeneous binding assay, comprising:
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a) preparing a plurality of amplification reagents, each comprising an amplification primer set having a first primer tagged with a peptidyl hapten tag and a second primer tagged with an affinity tag, wherein each pair of said first primers and said second primers are complementary to flanking sequences of a plurality of nucleic acid target sequences to be assayed; b) preparing a plurality of paramagnetic bead reagents, each comprising a paramagnetic microbead with a binding agent, wherein each of said binding agents has a binding affinity for one of said affinity tags of said second primers; c) preparing a plurality of test pads in a test pad area, each test pad having a different immobilized capture antibody having a binding affinity for one of said peptidyl hapten tags of said first primers; d) processing a biological sample to release a nucleic acid fraction; e) contacting said nucleic acid fraction with said amplification reagents; f) performing an amplification to yield amplification products; and g) assaying said amplification products for two-tailed amplicons labeled with one of said peptidyl hapten tags of said first primers on a first end and one of said affinity tags of said second primers on a second end by; i) contacting said amplification products with said paramagnetic bead reagents, thereby binding said affinity tags of said second ends of said two-tailed amplicons to said binding agents of said paramagnetic bead reagents to yield two-tailed amplicon paramagnetic bead complexes; ii) sweeping said two-tailed amplicon paramagnetic bead complexes into close contact with said plurality of test pads by moving a magnetic force field from outside to inside said test pad area, thereby binding said peptidyl hapten tags of said first ends of said two-tailed amplicon of said two-tailed amplicon paramagnetic bead complex to said capture antibodies of said test pads to yield immobilized paramagnetic reporter complexes; iii) sweeping from said test pad area any unbound two-tailed amplicon paramagnetic bead complexes by moving said magnetic force field from inside to outside said test pad area; and iv) detecting the presence of said immunoimmobilized paramagnetic reporter complexes on each of said test pads. - View Dependent Claims (52)
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53. A molecular detection complex comprising a two-tailed amplicon having a first end and a second end, said first end comprising a first primer covalently conjugated with a first peptidyl hapten tag, and said second end comprising a second primer covalently conjugated with a second peptidyl hapten tag, wherein said first end further comprises a first peptidyl hapten-bound anti-first peptidyl hapten antibody immobilized on a solid phase, and wherein said second end further comprises a second peptidyl hapten-bound anti-second peptidyl hapten antibody-coated reporter group.
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54. A method for rapid bioassay comprising:
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a) preparing an amplification primer set comprising a first primer comprising a first peptidyl hapten tag and a second primer comprising a second peptidyl hapten tag; b) forming a two-tailed amplicon product comprising said first peptidyl hapten tag of said first primer on a first end and said second peptidyl hapten tag of said second primer on a second end by amplifying a nucleic acid target with said amplification primer set; c) complexing said two-tailed amplicon product to a reporter group having a binding affinity for said second peptidyl hapten tag of said second primer to yield a two-tailed amplicon reporter group complex; d) capturing said two-tailed amplicon reporter group complex on a solid phase having an immobilized capture antibody, wherein said capture antibody has a binding affinity for said first peptidyl hapten tag, to yield an immobilized reporter group complex; and e) detecting said immobilized reporter group complex.
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55. A kit for performing a multiplex nucleic acid bioassay with multiplex detection of two-tailed amplicons, said kit comprising:
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a first assay reagent having a peptidyl-hapten conjugated first amplification primer; a plurality of test pads, each of said test pad comprising a dehydrated first affinity binding agent with affinity for said peptidyl-hapten of said first primer; and a second assay reagent comprising a bead reagent as a reporter group, wherein said bead reagent is coated with a second affinity binding agent. - View Dependent Claims (56)
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Specification