Saturation Assay

• US 20100062544A1
• Filed: 11/09/2007
• Published: 03/11/2010
• Est. Priority Date: 11/10/2006
• Status: Active Grant

• Current Assignee: Platform Diagnostics Limited
• Original Assignee: Platform Diagnostics Limited
• Inventors: Allen, Gerald John, Ruddell, Carolyn Jennifer, Evans, Douglas Robert
• Granted Patent: US 9,028,771 B2
• Time in Patent Office: Days
• US Class Current: 436/518
• CPC Class Codes: B01D 15/34 Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0825 Test strips
• CPC Class Codes: B01L 3/5023 with a sample being transported to, and subsequently stored in an absorbent for analysis
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: G01N 33/538 by sorbent column, particles or resin strip , i.e. sorbent materials
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction

Abstract

The invention provides a modified form of saturation assay, which is based on the measurement of a free or unbound labelled reagent fraction (such that there is an increase in signal in the presence of analyte) and employs trapping zones to concentrate said unbound or free labelled fraction to avoid loss of sensitivity. Preferably, the assay is a membrane assay.

Claims

1. A method of performing a saturation assay for detecting an analyte in a sample, wherein the method comprises a step in which a free or unbound fraction of labelled reagent resulting from a competitive binding event becomes concentrated at a designated trapping zone.

2. A method according to claim 1, wherein the saturation assay is an immunoassay comprising the steps of:

a. competitive binding of the analyte and an unlabelled analyte analogue to a labelled analyte binding reagent;

b. separation of the fraction of labelled analyte binding reagent bound to analyte analogue from the free fraction of labelled analyte binding reagent which is not so bound;

c. immobilisation and concentration of the free fraction of labelled analyte binding reagent; and

d. detection of immobilised and concentrated free fraction of labelled analyte binding reagent.

3. A method according to claim 1, wherein the saturation immunoassay is an agglutination assay comprising the steps of:

a. competitive binding of the analyte and an unlabelled multivalent analyte analogue to a labelled analyte-binding reagent;

b. separation of agglutinated and non-agglutinated fractions of labelled analyte-binding reagent;

c. immobilisation and concentration of the non-agglutinated fraction of labelled analyte-binding reagent; and

d. detection of immobilised and concentrated labelled analyte-binding reagent.

4. A method according to claim 1, wherein the saturation immunoassay is an immunoassay comprising the steps of:

a. competitive binding of the analyte and a labelled analyte analogue to an unlabelled analyte-binding reagent;

b. separation of the fraction of labelled analyte analogue bound to the analyte-binding reagent from the free fraction of labelled analyte analogue which is not so bound;

c. immobilisation and concentration of the free fraction of labelled analyte analogue; and

d. detection of immobilised and concentrated free fraction of labelled analyte analogue.

5. A method according to claim 1, wherein the saturation immunoassay is an agglutination assay comprising the steps of:

a. competitive binding of the analyte and a labelled multivalent analyte analogue to an unlabelled analyte-binding reagent;

b. separation of agglutinated and non-agglutinated fractions of labelled multivalent analyte analogue;

c. immobilisation and concentration of the non-agglutinated fraction of labelled multivalent analyte analogue; and

d. detection of immobilised and concentrated non-agglutinated fractions of labelled multivalent analyte analogue.

6. A method according to claim 3 wherein agglutinated and non-agglutinated fractions are separated by means whereby the agglutinated fraction becomes immobilised.

7. A method according to claim 6 wherein the agglutinated fraction becomes immobilised by means of a primary trapping zone comprising a capillary membrane of a pore size that excludes the agglutinated fraction.

8. A method according to claim 1, wherein the bound or agglutinated fraction becomes immobilised by an immobilised analyte-binding reagent or other ligand.

9. A method according to claim 6 wherein the agglutinated and non-agglutinated fractions are separated chromatographically by means of solvent flow through a porous membrane.

10. A method according to claim 1 wherein the unbound free fraction of labelled binding reagent is immobilised and concentrated by means of a secondary trapping zone comprising a capillary membrane of a pore size that excludes the labelled reagent.

12. A method according claim 1 wherein the unbound free fraction of labelled binding reagent is immobilised and concentrated by means of a secondary trapping zone comprising an immobilised analyte-binding reagent or ligand or other cognate binding partner.

13. A method according to claim 12 wherein the unbound free fraction of labelled binding reagent immobilised and concentrated by means of a secondary trapping zone provides a visually detectable indication.

11. (canceled)

14. (canceled)

15. A kit for performing a saturation assay for detection of an analyte in a sample, the kit comprising:

a. means for separating bound and unbound products of a competitive binding step; and

b. a secondary trapping zone.

16. A kit according to claim 15 further comprising a porous carrier

17. A kit according to claim 15, wherein the means for separating bound and unbound products of a competitive binding step comprises a primary trapping zone.

18. A kit according to claim 15, comprising a detectable moiety that is biological or non-biological.

21. A kit according to claim 15, wherein the saturation assay is an agglutination immunoassay and the primary trapping zone comprises a means for immobilising an agglutinated fraction.

22. A kit according to claim 15, wherein the detectable moiety is agglutinable.

23. A kit according to claim 21, wherein the primary trapping zone comprises a capillary membrane of a pore size that excludes the agglutinated fraction.

24. A kit according to claim 15, wherein the means for separating bound and unbound products of a competitive binding step comprises a capillary membrane of a pore size that allows chromatographic separation of bound and unbound products on the basis of particle size.

25. A kit according to claim 15, wherein the secondary trapping zone comprises means to immobilise and concentrate a free fraction of labelled analyte-binding reagent

26. A kit according to claim 25, wherein the secondary trapping zone comprises a capillary membrane of a pore size that excludes unagglutinated labelled reagent.

27. A kit according to claim 26, wherein the secondary trapping zone comprises an immobilised analyte-binding reagent or ligand or other cognate binding partner.

28. A kit according to claim 15, further comprising:

a. a hub to which two or more analyte-analogue moieties are bound; and

b. two or more labelled analyte-binding reagents capable of binding said analyte-analogue moieties

29. A kit according to claim 15, further comprising:

a. a hub to which two or more analyte-binding reagents are bound; and

b. a signal particle comprising two or more analyte-analogue moieties capable of binding said analyte-binding reagents.

33. A kit according to claim 28, wherein the pore size upstream of the primary trapping zone is sufficient to allow free movement of the hub, labeled reagent, sample and any agglutinate.

19-20. -20. (canceled)

30-32. -32. (canceled)

34. A device for performing a saturation agglutination assay for detection of an analyte within a sample, the device comprising a carrier having a proximal end for receiving a sample, and a distal end toward which a sample may travel along the carrier, wherein the carrier comprises:

a. a primary trapping zone comprising means which exclude an agglutinated fraction, and

b. a secondary trapping zone comprising means which exclude a labelled reagent.

35. A device according to claim 34 wherein the carrier is porous.

36. A device according to claim 35 wherein the primary and/or secondary trapping zones comprise a capillary membrane of a pore size that excludes an agglutinated fraction or labelled reagent, respectively.

37-38. -38. (canceled)