Method of adjusting the working range of a multi-analyte assay
First Claim
1. A method of quantitatively determining the concentration of at least one of a plurality of analytes in a fluid sample, said method comprising:
- a) providing an assay system for quantitatively determining the concentration of a plurality of analytes in a fluid sample, said assay system comprising at least one capture agent immobilized on a solid support, and 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 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 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, wherein said adjusting step comprises;
i) 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
ii) introducing said scaling agent (S) to said sample to create a reaction mixture and allowing the reaction mixture to come to equilibrium, whereby 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 ([Af]), and a free scaling agent portion having a free scaling agent concentration ([Sf]);
wherein said equilibrium reaction mixture comprises a total scaling agent concentration ([ST]) equal to the sum of said free scaling agent concentration plus said scaling agent-analyte complex concentration ([Sf]+[AS]), and a total analyte concentration ([AT]) equal to the sum of said free analyte concentration plus said scaling agent-analyte complex concentration ([Af]+[AS]), wherein said scaling agent (S) binds said high concentration analyte with a binding affinity Ka, [ST] is greater than [Ar], and the scaling constant, α
, is a coefficient proportional to Ka[ST]+1;
d) subsequent to the reaction mixture coming to equilibrium, introducing at least said free analyte portion of said reaction mixture to a solid support comprising at least one immobilized capture agent, said at least one immobilized capture agent having binding specificity for said high concentration analyte, under conditions permitting binding between said high concentration analyte 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 high concentration analyte in said fluid sample.
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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.
58 Citations
19 Claims
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1. A method of quantitatively determining the concentration of at least one of a plurality of analytes in a fluid sample, said method comprising:
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a) providing an assay system for quantitatively determining the concentration of a plurality of analytes in a fluid sample, said assay system comprising at least one capture agent immobilized on a solid support, and 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 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 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, wherein said adjusting step comprises;i) 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 ii) introducing said scaling agent (S) to said sample to create a reaction mixture and allowing the reaction mixture to come to equilibrium, whereby 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 ([Af]), and a free scaling agent portion having a free scaling agent concentration ([Sf]);
wherein said equilibrium reaction mixture comprises a total scaling agent concentration ([ST]) equal to the sum of said free scaling agent concentration plus said scaling agent-analyte complex concentration ([Sf]+[AS]), and a total analyte concentration ([AT]) equal to the sum of said free analyte concentration plus said scaling agent-analyte complex concentration ([Af]+[AS]), wherein said scaling agent (S) binds said high concentration analyte with a binding affinity Ka, [ST] is greater than [Ar], and the scaling constant, α
, is a coefficient proportional to Ka[ST]+1;d) subsequent to the reaction mixture coming to equilibrium, introducing at least said free analyte portion of said reaction mixture to a solid support comprising at least one immobilized capture agent, said at least one immobilized capture agent having binding specificity for said high concentration analyte, under conditions permitting binding between said high concentration analyte 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 high concentration analyte in said fluid sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification