Methods for detecting assay interferents and increasing dynamic range
The disclosure provides kits and methods for detecting a substance that interferes with detection of an analyte in a sample and for expanding the dynamic range and reducing the hook effect of an immunoassay. The kits and methods employ two conjugates with two different detectable labels, at least one of which is a chemiluminescent compound of Formula (I).
1 . A kit comprising:
(a) a first conjugate comprising a first detectable label attached to a first specific binding member that specifically binds an analyte,
(b) a second conjugate comprising a second detectable label attached to a second specific binding member, wherein the second specific binding member specifically binds a substance which interferes with detection of the analyte in a sample,
(c) a third specific binding member attached to a solid support, which either concurrently or competitively binds to the analyte and the substance which interferes with detection of the analyte, and optionally
(d) a fourth specific binding member attached to the solid support, which specifically binds to the substance which interferes with detection of the analyte, wherein at least one of the first and second detectable labels comprises a compound of formula (I), or a salt thereof:
wherein:
X is ethylenediamine, 4,7,10-trioxa-1,13-tridecanediamine,
Y is nitrogen, oxygen, or sulfur,
wherein when Y is nitrogen, R 1 is —SO 2 -A, wherein A is phenyl that is unsubstituted or substituted with 1 or 2 substituents selected from C 1 -C 4 alkyl, and when Y is oxygen or sulfur, R 1 is absent;
Q is —SO 2 — or —CO—;
L 1 and L 2 are each independently a C 1 -C 4 alkylene;
R 2 is —COOZ;
Z is hydrogen or C 1 -C 4 alkyl; and
R a , R b , R c , R d , R e , R f , R g , and R h are each hydrogen.
2 . The kit of claim 1 , wherein the second specific binding member preferentially binds to the substance that interferes with detection of the analyte in the presence of the analyte.
3 . The kit of claim 1 , wherein the analyte is an antigen and the substance that interferes with detection of the analyte is an auto-antibody that binds to the antigen in a sample.
4 . The kit of claim 1 , wherein the substance that interferes with detection of the analyte is biotin.
5 . A kit comprising:
(a) a first specific binding member comprising a biotin molecule and a first detectable label attached thereto, wherein the first specific binding member specifically binds an analyte;
(b) a conjugate comprising a second detectable label attached to a second specific binding member that specifically binds to the analyte;
(c) a solid support coated with streptavidin, wherein the streptavidin binds to the biotin molecule attached to the first specific binding member and a biotin molecule which interferes with detection of the analyte in a sample,
wherein at least one of the first and second detectable labels comprises a compound of formula (I), or a salt thereof:
wherein:
X is ethylenediamine, 4,7,10-trioxa-1,13-tridecanediamine,
Y is nitrogen, oxygen, or sulfur,
wherein when Y is nitrogen, R 1 is —SO 2 -A, wherein A is phenyl that is unsubstituted or substituted with 1 or 2 substituents selected from C 1 -C 4 alkyl, and when Y is oxygen or sulfur, R 1 is absent;
Q is —SO 2 — or —CO—;
L 1 and L 2 are each independently a C 1 -C 4 alkylene;
R 2 is —COOZ;
Z is hydrogen or C 1 -C 4 alkyl; and
R a , R b , R c , R d , R e , R f , R g , and R h are each hydrogen.
6 . A kit comprising:
(a) a first conjugate comprising a first detectable label attached to a first specific binding member that binds an analyte,
(b) a second conjugate comprising a second detectable label attached to a second specific binding member, wherein the second specific binding member binds the same analyte as the first specific binding member and the binding affinity of the first specific binding member for the analyte is greater than that of the second specific binding member, and
(c) a third specific binding member attached to a solid support, which can bind to the analyte concurrently with either the first or second specific binding member, wherein at least one of the first and second detectable labels comprises a compound of formula (I), or a salt thereof:
wherein:
X is ethylenediamine, 4,7,10-trioxa-1,13-tridecanediamine,
Y is nitrogen, oxygen, or sulfur,
wherein when Y is nitrogen, R 1 is —SO 2 -A, wherein A is phenyl that is unsubstituted or substituted with 1 or 2 substituents selected from C 1 -C 4 alkyl, and when Y is oxygen or sulfur, R 1 is absent;
Q is —SO 2 — or —CO—;
L 1 and L 2 are each independently a C 1 -C 4 alkylene;
R 2 is —COOZ;
Z is hydrogen or C 1 -C 4 alkyl; and
R a , R b , R c , R d , R e , R f , R g , and R h are each hydrogen.
7 . The kit of claim 6 , wherein the difference in binding affinity of the first specific binding member and the second specific binding member for the analyte is from about 5-fold to about 100-fold.
8 . A kit comprising:
(a) a first conjugate comprising a first specific binding member that binds an analyte and a first detectable label;
(b) a second conjugate comprising a second specific binding member and a second detectable label, wherein (i) the first specific binding member and second specific binding member are the same or are different, and (ii) the first and second detectable labels are different;
(c) a third specific binding member attached to a solid support, which can bind to the analyte concurrently with either the first or second specific binding member, wherein at least one of the first and second detectable labels comprises a compound of formula (I), or a salt thereof:
wherein:
X is ethylenediamine, 4,7,10-trioxa-1,13-tridecanediamine,
Y is nitrogen, oxygen, or sulfur,
wherein when Y is nitrogen, R 1 is —SO 2 -A, wherein A is phenyl that is unsubstituted or substituted with 1 or 2 substituents selected from C 1 -C 4 alkyl, and when Y is oxygen or sulfur, R 1 is absent;
Q is —SO 2 — or —CO—;
L 1 and L 2 are each independently a C 1 -C 4 alkylene;
R 2 is —COOZ;
Z is hydrogen or C 1 -C 4 alkyl; and
R a , R b , R c , R d , R e , R f , R g , and R h are each hydrogen.
9 . The kit of claim 1 , wherein X in the compound of formula (I) is
10 . The kit of claim 1 , wherein the compound has formula (Ia):
wherein:
each R is independently C 1 -C 4 alkyl;
m is 0, 1, or 2; and
n is 2, 3, or 4.
11 . The kit of claim 1 , wherein the compound has formula (Ib):
12 . The kit of claim 1 , wherein the first and second conjugates are of Formula (II):
wherein:
X is ethylenediamine, 4,7,10-trioxa-1,13-tridecanediamine,
Y is nitrogen, R 1 is —SO 2 -A, wherein A is phenyl that is unsubstituted or substituted with 1 or 2 substituents selected from C 1 -C 4 alkyl, and when Y is oxygen or sulfur, R 1 is absent;
Q is —SO 2 — or —CO—;
L 1 is a C 1 -C 4 alkylene;
L 3 is a C 1 -C 4 alkylene linker;
R a , R b , R c , R d , R e , R f , R g , and R h are each hydrogen, and
the binding member is a molecule capable of binding to a target analyte.
13 . The kit of claim 1 , wherein the fluorophore is fluorescein, a rhodamine, a boron-dipyrromethene, a cyanine, an oxazine, a thiazine, a coumarin, a naphthalimide, a rhodol, a naphthalene, a squaraine, a porphyrin, a flavin, or a lanthanide-based dye.
14 . The kit of claim 1 , wherein the fluorophore is
15 . The kit of claim 1 , wherein the first, second, third, and/or fourth specific binding member is an antibody or an antigen-binding fragment thereof.
16 . The kit of claim 1 , wherein the first and second specific binding members are directly attached to the first and second detectable labels, respectively.
17 . The kit of claim 1 , wherein the solid support is a particle, a microparticle, a bead, an electrode, or a multiwell plate.
18 . The kit of claim 17 , wherein the solid support comprises two or more spatially separated electrodes.
19 . The kit of claim 1 , wherein the first and second detectable labels are different.
20 . A method of detecting a substance that interferes with detection of an analyte in a sample, which method comprises:
(a) contacting a sample suspected of comprising an analyte and a substance that interferes with detection of the analyte with the kit of claim 1 under conditions that allow:
(i) binding of the analyte to the third specific binding member,
(ii) specific binding of the substance that interferes with detection of the analyte to the third specific binding member, or non-specific binding of the substance that interferes with detection of the analyte to the solid support surface,
(iii) binding of the first conjugate to the analyte,
(iv) binding of the second conjugate to the substance that interferes with detection of the analyte; and optionally
(v) binding of a fourth specific binding member to the substance that interferes with detection of the analyte;
(b) detecting the signal intensities of the first detectable label and the second detectable label; and
(c) detecting the presence of the substance that interferes with detection of the analyte in the sample by quantifying and analyzing the signal intensities of the first detectable label and the second detectable label.
21 . A method of detecting a substance that interferes with detection of an analyte in a sample, which method comprises:
(a) contacting a sample suspected of comprising an analyte and a substance that interferes with detection of the analyte with the kit of claim 1 under conditions that allow:
(i) binding of the analyte to the third specific binding member and binding of the analyte to the substance that interferes with detection of the analyte to form an analyte-interferent complex,
(ii) binding of the first conjugate to the analyte, and
(iv) binding of the second conjugate to the substance that interferes with detection of the analyte or the analyte-interferent complex; and optionally
(v) binding of a fourth specific binding member to the substance that interferes with detection of the analyte;
(b) detecting the signal intensities of the first detectable label and the second detectable label; and
(c) detecting the presence of the substance that interferes with detection of the analyte in the sample by quantifying and analyzing the signal intensities of the first detectable label and the second detectable label.
22 . A method of detecting a substance that interferes with detection of an analyte in a sample, which method comprises:
(a) contacting a sample suspected of comprising an analyte and a substance that interferes with detection of the analyte with the kit of claim 1 under conditions that allow:
(i) binding of the analyte to the third specific binding member,
(ii) binding of the first conjugate to the analyte and binding of the first conjugate to the substance that interferes with detection of the analyte, and
(iv) binding of the second conjugate to the substance that interferes with detection of the analyte; and optionally
(v) binding of a fourth specific binding member to the substance that interferes with detection of the analyte;
(b) detecting the signal intensities of the first detectable label and the second detectable label; and
(c) detecting the presence of the substance that interferes with detection of the analyte in the sample by quantifying and analyzing the signal intensities of the first detectable label and the second detectable label.
23 . A method of detecting a substance that interferes with detection of an analyte in a sample, which method comprises:
(a) contacting a sample suspected of comprising an analyte and a substance that interferes with detection of the analyte with the kit of claim 1 under conditions that allow:
(i) binding of the analyte to the third specific binding member;
(ii) specific or non-specific binding of the substance that interferes with detection of the analyte to the solid support surface,
(iii) binding of the first conjugate to the analyte and specific or non-specific binding of the substance that interferes with detection of the analyte to the first conjugate, thereby increasing the amount of first conjugate available for detection,
(iv) binding of the second conjugate to the substance that interferes with detection of the analyte; and
(v) binding of a fourth specific binding member to the substance that interferes with detection of the analyte;
(b) detecting the signal intensities of the first detectable label and the second detectable label; and
(c) detecting the presence of the substance that interferes with detection of the analyte in the sample by quantifying and analyzing the signal intensities of the first detectable label and the second detectable label.
24 . A method for detecting a biotin molecule that interferes with detection of an analyte in a sample using the kit of claim 5 , which method comprises:
(a) establishing a standard signal intensity (R value) of the first detectable label binding to the streptavidin-coated solid support in the absence of an interfering biotin molecule;
(b) contacting a sample suspected of comprising both an analyte and a biotin molecule that interferes with detection of the analyte with the kit of claim 5 under conditions that allow:
(i) binding of the analyte to the first specific binding member to form a first complex;
(ii) binding of the conjugate to the analyte bound to the first specific binding member to form immunocomplexes; and
(iii) binding of the streptavidin-coated solid support to the biotin molecule attached to the first specific binding member and the biotin molecule which interferes with detection of the analyte in a sample;
(c) detecting the signal intensities of the first detectable label and the second detectable label;
(d) correcting for the signal intensity of the second detectable label; and
(e) detecting the presence of the biotin molecule that interferes with detection of the analyte in the sample.
25 . A method of expanding the dynamic range of an immunoassay, which method comprises:
(a) contacting a test sample suspected of comprising an analyte with the kit of claim 6 , wherein the analyte binds to the third specific binding member;
(b) removing analyte not bound to the third specific binding member by washing;
(c) binding the first conjugate to the analyte and the second conjugate to the analyte, wherein the first and second conjugates do not concurrently bind to the analyte;
(d) removing first and second conjugates not bound to the analyte by washing;
(b) measuring the signal intensities of the first detectable label and the second detectable label; and
(c) determining the concentration of the analyte by comparing the signal intensities of the first detectable label and the second detectable label based on a flag value, whereby the dynamic range of the immunoassay is expanded.
26 . A method of reducing hook effect and expanding the dynamic range of an immunoassay, which method comprises:
(a) contacting a test sample suspected of comprising an analyte with the kit of claim 8 , wherein the analyte binds to the third specific binding member and the first conjugate binds to the analyte;
(b) removing any unbound analyte and unbound first conjugate by washing;
(c) binding the second conjugate to the analyte, wherein the first and second conjugates do not concurrently bind to the analyte,
(d) removing any unbound second conjugate by washing;
(b) measuring the signal intensities of the first detectable label and the second detectable label; and
(c) determining the concentration of the analyte based on a flag value, whereby hook effect of the immunoassay is reduced and dynamic range is expanded.