Soluble analyte detection and amplification

• US 20050048500A1
• Filed: 09/02/2003
• Published: 03/03/2005
• Est. Priority Date: 09/02/2003
• Status: Active Grant

• Current Assignee: Restalyst Pte., Ltd.
• Original Assignee: New England Rare Reagents, Inc.
• Inventors: Lawton, Robert L.
• Granted Patent: US 7,341,837 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte

Abstract

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.

Claims

7-1. The method of claim 7, wherein said amplification of said nucleic acid portion comprises a polymerase chain reaction.

22. A method for increasing the sensitivity of solution-phase detection of a compound of interest, comprising the steps of:

a) providing a sample suspected of containing said compound of interest;

b) providing a binding construct comprising;

i) a recognition portion capable of binding said compound of interest, and

ii) a nucleic acid portion

c) contacting said sample with said binding construct for a period of time sufficient to permit said recognition portion to bind said compound of interest present in said sample, thereby forming construct-compound complexes in solution;

d) providing one or more surface, wherein said one or more surfaces bears one or more accessible binding target capable of binding to said recognition portion;

e) contacting said one or more surfaces with said solution for a period of time sufficient for said one or more accessible binding target to bind said recognition portion of any binding construct not bound to said compound of interest, thereby forming 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 binding construct in said solution,

wherein said separation of said construct-surface complexes from said solution results in a separation of substantially all binding constructs not bound to a compound of interest and in an increased sensitivity of detection of said compound of interest, and

wherein the presence of said nucleic acid portion of said binding construct indicates the presence of said compound of interest in said sample.

23. The method of claim 22, 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.

24. The method of claim 22, wherein said one or more surfaces comprises particles.

25. The method of claim 24, wherein said particles comprise magnetic particles.

26. The method of claim 25, wherein said step (f) comprises separating substantially all said construct-surface complexes from said solution by means of a magnet.

27. The method of claim 22, 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, hybridization of said nucleic acid portion, enzymatic amplification, detection of a label, or a combination thereof.

28. The method of claim 22, 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.

29. The method of claim 28, wherein said amplification of said nucleic acid portion comprises a polymerase chain reaction.

30. The method of claim 26, 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, hybridization of said nucleic acid portion, enzymatic amplification, detection of a label, or a combination thereof.

31. The method of claim 26, 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.

32. The method of claim 31, wherein said amplification of said nucleic acid portion comprises a polymerase chain reaction.

33. The method of claim 22, wherein said recognition portion comprises a receptor.

34. The method of claim 22, wherein said recognition portion comprises an antigen.

35. The method of claim 22, wherein said recognition portion comprises an antibody or antibody fragment.

36. The method of claim 22, wherein said recognition portion comprises a single chain antibody variable region fragment.

37. The method of claim 22, wherein said recognition portion comprises a Fab fragment.

38-2. The method of claim 21, wherein said nucleic acid portion comprises DNA.

39-3. The method of claim 21, wherein said nucleic acid portion comprises RNA.

42. A kit for detecting a compound of interest in a sample suspected of containing said compound of interest comprising:

a) a binding construct comprising

(i) a recognition portion which recognizes and binds said compound of interest, and

(ii) a nucleic acid portion; and

b) one or more surfaces bearing one or more accessible binding targets capable of binding to said recognition portion of said binding construct.

43. The kit of claim 42, further comprising a nucleic acid amplification primer pair, wherein each primer of said primer pair is capable of hybridizing to its complementary sequence at the 3′

end of a target nucleic acid sequence of said nucleic acid portion.