Rapid magnetic flow assays

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.

1 - 4 . (canceled) 
     
     
         5 . A method for multiplex target nucleic acid detection by heterogeneous binding assay, comprising:
 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. 
   
     
     
         6 . The method of  claim 5  wherein said ligand tag of said amplification reagent is biotin and said binding agent of said paramagnetic bead reagent is an avidin. 
     
     
         7 . The method of  claim 5  wherein said peptidyl hapten tag is a peptide with 2 to 100 amino acid residues. 
     
     
         8 . The method of  claim 5  wherein said immunoimmobilized paramagnetic complex on said test pad is detected visually. 
     
     
         9 . The method of  claim 5  wherein said amplification step comprises a thermocycling protocol or isothermal protocol. 
     
     
         10 . The method of  claim 5  wherein said amplification step further comprises reverse transcription. 
     
     
         11 . The method of  claim 5  wherein said amplification step comprises nested amplification. 
     
     
         12 . The method of  claim 5  further comprising a multiplex target detection protocol. 
     
     
         13 . A kit for performing the method of  claim 5 , said kit comprising said amplification reagent and said test pad area. 
     
     
         14 - 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. 
     
     
         35 . The molecular detection complex of  claim 34 , wherein said ligand-bound ligand binding agent-coated reporter group is a magnetic microbead coated with said ligand binding agent, and wherein said solid phase is a test pad area. 
     
     
         36 . An apparatus for forming and purifying the molecular detection complex of  claim 35 , comprising:
 a) a microfluidic cartridge comprising a substrate and a microchannel in said substrate, said microchannel comprising a fluid path with axis of flow and with upper and lower aspects;   b) a test pad in said microchannel, said test pad comprising an affinity capture agent comprising an anti-peptidyl hapten antibody immobilized on a solid phase;   c) a means for introducing a fluid comprising (i) a paramagnetic microbead comprising said reporter group and (ii) a two-tailed amplicon into said microchannel; and   d) a means for moving a magnetic force field along a plane parallel to said axis of flow of said microfluidic channel to (i) sweep said paramagnetic microbead in said fluid into close contact with said affinity capture agent, thereby binding said paramagnetic microbead to said affinity capture agent and binding said two-tailed amplicon to said paramagnetic microbead to yield said molecular detection complex, and (ii) sweep from said test pad any unbound paramagnetic microbead and any unbound two-tailed amplicon.   
     
     
         37 - 43 . (canceled) 
     
     
         44 . A method for rapid bioassay comprising:
 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.   
     
     
         45 . The method of  claim 44  wherein said reporter group is a magnetic microbead and said solid phase is a test pad. 
     
     
         46 . The method of  claim 44  wherein said reporter group is a fluorophore and said solid phase is a barcoded latex bead. 
     
     
         47 . The method of  claim 44 , wherein said steps a) through c) are performed for a plurality of first primers conjugated with different peptidyl hapten tags and a plurality of two-tailed amplicon products are captured and detected on a plurality of solid phases, each with an antibody specific for one of said different peptidyl hapten tags. 
     
     
         48 . The method of  claim 45 , wherein said steps a) through c) are performed for a plurality of first primers conjugated with different peptidyl hapten tags and a plurality of two-tailed amplicon products are magnetically captured and detected on a plurality of solid test pads, each with an antibody specific for one of said different peptidyl hapten tags. 
     
     
         49 . The method for multiplex target nucleic acid detection by heterogeneous binding assay of  claim 12 , wherein said amplification reagent comprises a plurality of first primers, each tagged with a different peptidyl hapten tag, and wherein said test pad area comprises a plurality of test pads, each with an immobilized monoclonal capture antibody having a binding affinity for one of the peptidyl hapten tags of the plurality of first primers. 
     
     
         50 . The method for multiplex target nucleic acid detection by heterogeneous binding assay of  claim 12 , wherein said test pad area comprises a plurality of test pads, each with an immobilized monoclonal capture antibody having a binding affinity for a different peptidyl hapten tagged amplicon. 
     
     
         51 . A method for multiplex target nucleic acid detection by heterogeneous binding assay, comprising:
 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. 
   
     
     
         52 . The method for multiplex target nucleic acid detection by heterogeneous binding assay of  claim 51 , wherein each of said affinity tags is a peptidyl hapten tag and each of said binding agents is an anti-peptidyl hapten antibody. 
     
     
         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. 
     
     
         54 . A method for rapid bioassay comprising:
 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.   
     
     
         55 . A kit for performing a multiplex nucleic acid bioassay with multiplex detection of two-tailed amplicons, said kit comprising:
 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.   
     
     
         56 . The kit of  claim 55  for performing a panel assay for multiplex detection of multiple nucleic acid targets.