Soluble analyte detection and amplification
View Patent ↗The present invention relates to a method of detection of a compound of interest that is present at low levels in a sample. In particular, the present invention relates to a method of detection of a compound of interest in solution by a nucleic acid-labelled binding construct, separation of the unbound nucleic acid-labelled binding construct, and the detection of the bound nucleic acid-labelled binding construct in the solution phase. The present invention is particularly adaptable to be used in conjunction with a nucleic acid amplification reaction for detecting the presence or absence of the nucleic acid portion of binding construct in a sample indicating the presence or absence of the compound of interest.
1. A method for detecting a non-nucleic acid compound of interest in a biological or environmental sample comprising the steps of:
a) providing a binding construct comprising an antibody or an antibody fragment portion which specifically recognizes and specifically binds said non-nucleic acid compound of interest without capture on a solid support, and a nucleic acid portion;
b) mixing said binding construct with said biological or environmental sample in a solution mixture to form construct-compound complexes in solution without capture on a solid support, wherein said non-nucleic acid compound of interest in said biological or environmental sample becomes specifically bound to said binding construct;
c) providing one or more surfaces, wherein said surfaces bear one or more accessible non-nucleic acid binding targets capable of specifically recognizing and specifically binding to said antibody or said antibody fragment portion of said binding construct;
d) introducing said one or more surfaces to said solution mixture of said construct-compound complexes after said non-nucleic acid compound of interest in said biological or environmental sample has become bound to said binding construct in order for said one or more surfaces to form construct-surface complexes in solution with any unbound binding constructs resulting in said solution mixture containing essentially said construct-compound complexes and said construct-surface complexes;
e) separating said construct-surface complexes from said solution mixture leaving behind said construct-compound complexes in solution; and
f) detecting the presence or absence of said nucleic acid portion of said construct-compound complexes in solution after said separation step (e), said detection being done without the presence of any of said surfaces in connection with said construct-compound complexes that are being detected;
wherein the presence of said nucleic acid portion of said construct-compound complexes indicates the presence of said non-nucleic acid compound of interest in said biological or environmental sample.
2. The method of claim 1 , wherein said one or more surfaces is selected from the group consisting of: particles, powders, beads, planar surfaces, non-planar surfaces, a tube, a well, non-porous films, non-porous membranes, porous films, porous membranes, fibers, fillers, meshes, grids, filters, matrices, gels, and combinations thereof.
3. The method of claim 1 , wherein said one or more surfaces comprise particles.
4. The method of claim 3 , wherein said particles comprise magnetic particles.
5. The method of claim 4 , wherein said step (e) comprises separating said construct-surface complexes out of said mixture by means of a magnet.
6. The method of claim 5 , wherein, in step (f), said detecting the presence or absence of said nucleic acid portion of said binding construct comprises amplification of said nucleic acid portion.
7. The method of claim 6 , wherein said amplification of said nucleic acid portion comprises a polymerase chain reaction.
8. The method of claim 1 , wherein, in step (f), said detecting the presence or absence of said nucleic acid portion of said binding construct comprises amplification of said nucleic acid portion.
9. The method of claim 8 , wherein said amplification of said nucleic acid portion comprises a polymerase chain reaction.
10. The method of claim 1 , wherein said antibody or antibody fragment portion comprises a single chain antibody variable region fragment.
11. The method of claim 1 , wherein said antibody or antibody fragment portion comprises a Fab fragment.
12. The method of claim 11 , wherein said Fab fragment is attached to said nucleic acid portion through the free sulfhydryl of the Fab fragment.
13. The method of claim 1 , wherein said nucleic acid portion comprises DNA.
14. The method of claim 1 , wherein said nucleic acid portion comprises RNA.
15. The method of claim 1 , wherein said nucleic acid portion comprises a nucleic sequence that does not include a sequence that is expected to be found in the biological or environmental sample.
16. The method of claim 1 , wherein said step (a) comprises providing two or more different types of binding constructs, wherein each of said two or more different binding constructs has a different antibody or antibody fragment portion and a different nucleic acid portion.
17. A method for solution-phase detection of a non-nucleic acid compound of interest, comprising the steps of:
a) providing a biological or environmental sample suspected of containing said non-nucleic acid compound of interest;
b) providing a binding construct comprising:
i) an antibody or an antibody fragment portion capable of specifically binding said non-nucleic acid compound of interest without capture on a solid support, and
ii) a nucleic acid portion
c) contacting said biological or environmental sample with said binding construct for a period of time sufficient to permit said antibody or antibody fragment portion to specifically bind said non-nucleic acid compound of interest present in said biological or environmental sample, thereby forming construct-compound complexes in solution, wherein said non-nucleic acid compound of interest in said biological or environmental sample becomes specifically bound to said binding construct;
d) providing one or more surfaces, wherein said one or more surfaces bears one or more accessible non-nucleic acid binding targets capable of specifically binding to said antibody or antibody fragment portion;
e) contacting said one or more surfaces with said solution alter said non-nucleic acid compound of interest in said biological or environmental sample has become bound to said binding construct for a period of time sufficient for said one or more accessible non-nucleic acid binding targets to specifically bind said antibody or antibody fragment portion of any binding construct not bound to said non-nucleic acid compound of interest, thereby forming construct-surface complexes in said solution resulting in said solution containing essentially said construct-compound complexes and said construct-surface complexes;
f) separating said construct-surface complexes from said solution, leaving said construct-compound complexes in said solution; and
g) detecting the presence or absence of said nucleic acid portion of said construct-compound complexes in said solution after said separation step (f), said detection being done without the presence of any said surfaces in connection with said construct-compound complexes that are being detected;
wherein said separation of said construct-surface complexes from said solution results in a separation of all binding constructs not bound to a non-nucleic acid compound of interest, and
wherein the presence of said nucleic acid portion of said construct-compound complexes indicates the presence of said non-nucleic acid compound of interest in said biological or environmental sample.
18. The method of claim 17 , wherein said one or more surfaces is selected from the group consisting of: particles, powders, beads, planar surfaces, non-planar surfaces, a tube, a well, non-porous films, non-porous membranes, porous films, porous membranes, fibers, fillers, meshes, grids, filters, matrices, gels, and combinations thereof.
19. The method of claim 17 , wherein said one or more surfaces comprise particles.
20. The method of claim 19 , wherein said particles comprise magnetic particles.
21. The method of claim 20 , wherein said step (f) comprises separating substantially all said construct-surface complexes from said solution by means of a magnet.
22. The method of claim 21 , wherein, in step (g), said detecting the presence or absence of said nucleic acid portion of said binding construct comprises amplification of said nucleic acid portion.
23. The method of claim 22 , wherein said amplification of said nucleic acid portion comprises a polymerase chain reaction.
24. The method of claim 17 , wherein, in step (g), said detecting the presence or absence of said nucleic acid portion of said binding construct comprises amplification of said nucleic acid portion.
25. The method of claim 24 , wherein said amplification of said nucleic acid portion comprises a polymerase chain reaction.
26. The method of claim 17 , wherein said antibody or antibody fragment portion comprises a single chain antibody variable region fragment.
27. The method of claim 17 , wherein said antibody or antibody fragment portion comprises a Fab fragment.
28. The method of claim 27 , wherein said Fab fragment is attached to said nucleic acid portion through the free sulfhydryl of the Fab fragment.
29. The method of claim 17 , wherein said nucleic acid portion comprises DNA.
30. The method of claim 17 , wherein said nucleic acid portion comprises RNA.
31. The method of claim 17 , wherein said nucleic acid portion comprises a nucleic sequence that does not include a sequence that is expected to be found in said biological or environmental sample.
32. The method of claim 17 , wherein said step (b) comprises providing two or more different types of binding constructs, wherein each of said two or more different binding constructs has a different antibody or antibody fragment portion and a different nucleic acid portion.