SOLUBLE ANALYTE DETECTION AND AMPLIFICATION

• US 20100184076A1
• Filed: 02/12/2010
• Published: 07/22/2010
• Est. Priority Date: 09/02/2003
• Status: Active Grant

• Current Assignee: Restalyst Pte., Ltd.
• Original Assignee: Restalyst Pte., Ltd.
• Inventors: Lawton, Robert L.
• Granted Patent: US 9,181,579 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

1. A method for detecting a compound of interest in a sample comprising the steps of:

a) providing a binding construct comprising a recognition portion which recognizes and binds said compound of interest, and a nucleic acid portion;

b) mixing said binding construct with said sample to form construct-compound complexes in solution;

c) providing one or more surfaces, wherein said surfaces are magnetic particles bearing one or more accessible binding targets capable of recognizing and binding to said recognition portion of said binding construct;

d) introducing said one or more surfaces to said solution of said binding construct and said sample in order for said one or more surfaces to form construct-surface complexes with any unbound binding constructs;

e) separating said construct-surface complexes from said solution leaving behind said construct-compound complexes in solution without physical removal of the construct-surface complexes from the solution; and

f) detecting the presence or absence of said nucleic acid portion of said binding construct in the solution,

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

2. 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, hybridization of said nucleic acid portion, enzymatic amplification, detection of a label, or a combination thereof.

3. 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.

4. The method of claim 3, wherein said amplification of said nucleic acid portion comprises a polymerase chain reaction.

5. The method of claim 1, wherein said recognition portion comprises a receptor.

6. The method of claim 1, wherein said recognition portion comprises an antigen.

7. The method of claim 1, wherein said recognition portion comprises an antibody or an antibody fragment.

8. The method of claim 1, wherein said recognition portion comprises a single chain antibody variable region fragment.

9. The method of claim 1, wherein said recognition portion comprises a Fab fragment.

10. The method of claim 9, wherein said Fab fragment is attached to said nucleic acid portion through the free sulfhydryl of the Fab fragment.

11. The method of claim 1 wherein said compound of interest comprises an antibody or antibody fragment, said recognition portion of said binding construct comprises an antigen that is recognized by said compound of interest, and said accessible binding targets comprise an antibody or antibody fragment that is capable of recognizing and binding to said recognition portion of said binding construct.

12. The method of claim 1, wherein said nucleic acid portion comprises DNA.

13. The method of claim 1, wherein said nucleic acid portion comprises RNA.

14. 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 sample.

15. 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 recognition portion and a different nucleic acid portion.