Method of adjusting the working range of a multi-analyte assay

• US 20060177873A1
• Filed: 09/06/2005
• Published: 08/10/2006
• Est. Priority Date: 02/04/2005
• Status: Active Grant

• Current Assignee: Courtagen Life Sciences, Inc.
• Original Assignee: Decision Biomarkers Incorporated (Courtagen Life Sciences, Inc.)
• Inventors: Dowd, Roger, Donahue, Jeffrey G.
• Granted Patent: US 7,396,689 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding

Abstract

The invention features a method of adjusting the concentration of at least one but not all of a plurality of analytes in a fluid sample to match a known working range of detection of an analyte assay system, where each of the plurality of analytes may or may not be present within an expected initial concentration range having a high end and a low end, and at least one analyte has a high end expected concentration range that exceeds the high end of the working range of the assay system. The expected concentration of the high concentration analyte is adjusted by a proportional scaling constant, α, so that the high end of the adjusted expected concentration range is less than or equal to the high end of the working range, without adjusting the expected concentration range of at least one other of the plurality of analytes. Adjustment is preferably accomplished by adding to the solution phase of the assay one or more scaling agents, each scaling agent binding with specificity to an analyte and thereby preventing it from being detected by the assay system, e.g., by competing with binding to immobilized capture agent. This scaling method contrasts with prior methods, in which a concentration of available analyte is offset by a fixed amount to adjust the detectable threshold of the assay. Here, the amount of scaling agent is proportional to a scaling coefficient, and the scaling agent is present in the solution phase of the assay at high concentrations relative to analyte. Due to the equilibrium conditions established by the laws of mass transfer, the amount of free analyte remaining in solution in the presence of scaling agent is predictable and finite, and can be measured as a quantitative indicator of the initial concentration of the analyte in the sample.

Claims

1. A method of adjusting the concentration of at least one but not all of a plurality of analytes in a fluid sample to match a working range of detection of an analyte assay system, said method comprising the steps of:

a) obtaining characterization of a working range of detection of an analyte assay system for quantitative determination of the concentration of a plurality of analytes in a fluid sample, said characterization comprising a high end and a low end of said working range;

b) providing a fluid sample in which each of a plurality of analytes may or may not be present within an expected initial concentration range having a high end and a low end, said plurality of analytes comprising at least one high concentration analyte having a high end expected concentration range that exceeds the high end of said working range of said assay system; and

c) adjusting the expected concentration of said high concentration analyte by a proportional scaling constant, α

, so that said high end of said adjusted expected concentration range is less than or equal to the high end of said working range, without adjusting the expected concentration range of at least one other of said plurality of analytes.

2. The method of claim 1, wherein a ratio of said adjusted concentration of said high concentration analyte to the initial concentration of said high concentration analyte is independent of said initial concentration of said high concentration analyte.

3. The method of claim 1, wherein said adjusting step comprises the steps of:

a) providing a scaling agent (S) having binding specificity for at least one, but not all, of said plurality of analytes, whereby binding of analyte by said scaling agent prevents detection of said analyte by said assay system; and

b) introducing a certain amount of said scaling agent (S) to said sample to create a reaction mixture, under conditions such that, subsequent to the reaction mixture coming substantially to equilibrium binding conditions, the reaction mixture comprises a scaling agent-analyte complex portion having a scaling agent-analyte complex concentration ([AS]), a free analyte portion having a free analyte concentration ([A_f]), and a free scaling agent portion having a free scaling agent concentration ([S_f]), wherein said introducing results in a total scaling agent concentration ([S_T]) in said reaction mixture equal to the sum of said free scaling agent concentration plus said scaling agent-analyte complex concentration ([S_f]+[AS]), and a total analyte concentration ([A_T]) in said reaction mixture equal to the sum of said free analyte concentration plus said scaling agent-analyte complex concentration ([A_f]+[AS]).

4. The method of claim 3, wherein [S_T] is greater than or equal to the high end expected initial concentration of said high concentration analyte in said reaction mixture.

5. The method of claim 3, wherein [S_T] is at least four times greater than [A_f] and the product of [A_f] and K_a is less than one fourth.

6. The method of claim 3, wherein [S_T] is at least nine times greater than [A_f] and the product of [A_f] and K_a is less than one ninth.

7. The method of claim 3, wherein said scaling agent is a dry reagent.

8. A method of quantitatively determining the concentration of a plurality of analytes in a fluid sample, said method comprising the steps of:

a) providing an assay system for the quantitative determination of the concentration of a plurality of analytes in a fluid sample, said assay system comprising a defined working range having a high end and a low end;

b) providing a fluid sample in which each of a plurality of analytes which may or may not be present within an expected initial concentration range having a high end and a low end, said plurality of analytes comprising at least one high concentration analyte having a high end expected concentration range that exceeds the high end of said working range of said assay system;

c) adjusting the expected concentration of said high concentration analyte by a proportional scaling constant, α

, so that said high end of said adjusted expected concentration range is less than or equal to the high end of said working range, without adjusting the expected concentration range of at least one other of said plurality of analytes;

d) introducing said sample to a solid support comprising at least one immobilized capture agent, said capture agent having binding specificity for at least one of said plurality of analytes, under conditions permitting binding between said analytes and said capture agent to form analyte-capture agent complex; and

e) determining said analyte-capture agent complex on said solid support as an indication of the initial concentration of said analytes in said fluid sample.

9. The method of claim 8, wherein said adjusting step precedes introducing said fluid sample to said solid support.

10. The method of claim 8, wherein introducing said fluid sample to said solid support precedes said adjusting step.

11. The method of claim 8, wherein said adjusting step coincides with introducing said fluid sample to said solid support.

12. The method of claim 8, wherein a ratio of said adjusted concentration of said high concentration analyte to the initial concentration of said high concentration analyte is independent of said initial concentration of said high concentration analyte.

13. The method of claim 8, wherein said adjusting step comprises the steps of:

a) providing a scaling agent (S) having binding specificity for at least one, but not all, of said plurality of analytes, wherein binding of said scaling agent to said high concentration analyte prevents binding of said high concentration analyte to said at least one capture agent; and

b) introducing a certain amount of said scaling agent (S) to said sample to create a reaction mixture, under conditions such that, subsequent to the reaction mixture coming substantially to equilibrium binding conditions, the reaction mixture comprises a scaling agent-analyte complex portion having a scaling agent-analyte complex concentration ([AS]), a free analyte portion having a free analyte concentration ([A_f]), and a free scaling agent portion having a free scaling agent concentration ([S_f]);

wherein said introducing results in a total scaling agent concentration ([S_T]) in said reaction mixture equal to the sum of said free scaling agent concentration plus said scaling agent-analyte complex concentration ([S_f]+[AS]), and a total analyte concentration ([A_T]) in said reaction mixture equal to the sum of said free analyte concentration plus said scaling agent-analyte complex concentration ([A_f]+[AS]).

14. The method of claim 13, wherein said total scaling agent concentration ([S_T]) is at least four times greater than said free analyte concentration ([A_f]), and the product of [A_f] and K_a is less than one fourth.

15. The method of claim 13, wherein said total scaling agent concentration ([S_T]) is at least nine times greater than said free analyte concentration ([A_f]), and the product of [A_f] and K_a is less than one ninth.

16. The method of claim 13, wherein said scaling agent (S) binds said high concentration analyte with a binding affinity K_a, and wherein [S_T] is greater than [A_T] and the scaling constant, α

, is proportional to (K_a[S_T]+1).

17. The method of claim 16, wherein said scaling constant, α

, equals (K_a [S_T]+1).

18. The method of claim 13, wherein [S_T] is greater than or equal to [A_T] and wherein a ratio of the free analyte concentration to the total analyte concentration, ([A_f][A_T]), is independent of said total analyte concentration ([A_T]) at said total scaling agent concentration ([S_T]).

19. The method of claim 13, wherein a ratio of free analyte concentration to total analyte concentration, ([A_f][A_T]), is independent of said total analyte concentration within the adjusted concentration range.

20. The method of claim 8, wherein said determining step comprises measuring a parameter representative of the amount of each analyte bound to said capture agent as an indication of the initial concentration of said analyte in said sample.

21. The method of claim 8, wherein said determining step comprises measuring a detectable signal.

22. The method of claim 13, wherein said determining said analyte-capture agent complex on said solid support comprises introducing at least one detection binder to said solid support under conditions permitting said detection binder to bind said analyte-capture agent complex, and determining the amount of detection binder bound to said analyte-capture agent complex as an indication of the initial concentration of said analytes in said fluid sample.

23. The method of claim 22, wherein said detection binder comprises a detectable signal, and wherein said determining the amount of detection binder bound to said analyte-capture agent complex comprises measuring said detectable signal, said signal being proportional to said concentration of said analyte in said sample.

24. The method of claim 23, wherein said method further comprises the step of removing said scaling agent-analyte complex from said reaction mixture prior to said contacting the reaction mixture with a solid support.

25. The method of claim 13, wherein binding of said scaling agent to said high concentration analyte prevents binding of said high concentration analyte to said at least one capture agent, and wherein said method further comprises the step of, prior to introducing said sample to said solid support, introducing to said reaction mixture an analyte analogue conjugate comprising a detectable signal, said analogue conjugate capable of competing with said high concentration analyte for binding to said scaling agent and said capture agent.

26. The method of claim 25, wherein said determining the amount of each of said analytes bound to said at least one capture agent comprises determining the amount of analyte analogue conjugate bound to said solid support as an inverse indication of the amount of analyte bound to said solid support, and as an inverse indication of the initial concentration of said analytes in said fluid sample.

27. The method of claim 26, wherein said analyte analogue conjugate is present in said reaction mixture at a total analyte analogue conjugate concentration [A*_T], and said total scaling agent concentration [S_T] is greater than or equal to a sum of said total analyte concentration and said total analyte analogue conjugate concentration ([A_T]+[A*_T]).

28. The method of claim 25, wherein said total scaling agent concentration ([S_T]) is at least four times greater than said free analyte concentration ([A_f]), and the product of [A_f] and K_a is less than one fourth.

29. The method of claim 25, wherein said total scaling agent concentration ([S_T]) is at least nine times greater than said free analyte concentration ([A_f]), and the product of [A_f] and K_a is less than one ninth.

30. A method of measuring the affinity of a binding agent, K_a, for an analyte, said method comprising the steps of:

a) providing a fluid sample comprising an analyte;

b) experimentally determining a value of a scaling constant α

; and

c) calculating K_a, where K_a is equal to (α

−

1)/[S_f].

31. An apparatus for performing an assay for the quantitative determination of the concentration of a plurality of analytes in a fluid sample, said assay system comprising a defined working range having a high end and a low end, said apparatus comprising;

a) means for providing a fluid sample in which each of a plurality of analytes which may or may not be present within an expected initial concentration range having a high end and a low end, said plurality of analytes comprising at least one high concentration analyte having a high end expected concentration range that exceeds the high end of said working range of said assay system;

b) means for adjusting the expected concentration of said high concentration analyte by a proportional scaling constant, α

, so that said high end of said adjusted expected concentration range is less than or equal to the high end of said working range, without adjusting the expected concentration range of at least one other of said plurality of analytes;

c) means for introducing said sample to a solid support comprising at least one immobilized capture agent, said capture agent having binding specificity for at least one of said plurality of analytes, under conditions permitting binding between said analytes and said capture agent to form analyte-capture agent complex; and

d) means for determining said analyte-capture agent complex on said solid support as an indication of the initial concentration of said analytes in said fluid sample.

32. The apparatus of claim 31, wherein said adjusting means comprise means for introducing a certain amount of said scaling agent (S) to said sample to create a reaction mixture, under conditions such that, subsequent to the reaction mixture coming substantially to equilibrium binding conditions, the reaction mixture comprises a scaling agent-analyte complex portion having a scaling agent-analyte complex concentration ([AS]), a free analyte portion having a free analyte concentration ([A_f]), and a free scaling agent portion having a free scaling agent concentration ([S_f]);

wherein said introducing results in a total scaling agent concentration ([S_T]) in said reaction mixture equal to the sum of said free scaling agent concentration plus said scaling agent-analyte complex concentration ([S_f]+[AS]), and a total analyte concentration ([A_T]) in said reaction mixture equal to the sum of said free analyte concentration plus said scaling agent-analyte complex concentration ([A_f]+[AS]) and wherein said scaling agent (S) having binding specificity for at least one, but not all, of said plurality of analytes.

33. The apparatus of claim 31, wherein said means for determining comprises means for measuring a parameter representative of the amount of each analyte bound to said capture agent as an indication of the initial concentration of said analyte in said sample.

34. The apparatus of claim 31, wherein said means for determining comprises means for measuring a detectable signal.

35. The apparatus of claim 31, wherein said means for determining comprises means for introducing at least one detection binder to said solid support under conditions permitting said detection binder to bind said analyte-capture agent complex, and means for determining the amount of detection binder bound to said analyte-capture agent complex as an indication of the initial concentration of said analytes in said fluid sample.

36. The apparatus of claim 35, wherein said means for determining the amount of detection binder bound to said analyte-capture agent complex comprises means for measuring said detectable signal, said signal being proportional to said concentration of said analyte in said sample.

37. The apparatus of claim 31, wherein said apparatus further comprises means for removing said scaling agent-analyte complex from said reaction mixture prior to said contacting the reaction mixture with a solid support.

38. The apparatus of claim 31, wherein said apparatus further comprises means for introducing to said reaction mixture an analyte analogue conjugate comprising a detectable signal, said analogue conjugate capable of competing with said high concentration analyte for binding to said scaling agent and said capture agent.

39. The apparatus of claim 38, wherein said apparatus comprises means for determining the amount of analyte analogue conjugate bound to said solid support as an inverse indication of the amount of analyte bound to said solid support, and as an inverse indication of the initial concentration of said analytes in said fluid sample.