Methods and compositions for detection and quantification of small molecules and other analytes

A method of detecting a target analyte in a liquid sample, the method comprising the steps of:

• (a) contacting a liquid sample containing or suspected of containing a target analyte with:
• (i) a plurality of detection probes that specifically bind to the target analyte, and
• (ii) a plurality of detectable moieties, the detectable moieties being linked to one or more immobilized target analytes, wherein the detection probes competitively bind to the target analytes contained in the liquid sample and to the immobilized target analytes;
• (b) incubating the product of step (a) to allow binding of the detection probes to the target analytes contained in the liquid sample or to the immobilized target analytes;
• (c) contacting the product of step (b) with a plurality of capture probes, the capture probes being linked to one or more capture ligands, wherein the capture ligand specifically binds to the detection probe, and incubating to allow capture ligands to bind to detection probes;
• (d) separating at least a portion of the capture probes from the liquid sample;
• and
• (e) detecting the detectable moieties that are associated with the capture probes of step (d), wherein detectable moieties are associated with capture probes by binding of a linked immobilized target analyte to a detection probe that is bound to a capture ligand linked to the capture probe, thereby detecting the target analyte in the liquid sample, wherein the concentration of the target analyte in the liquid sample is inversely proportional to a signal of the detectable moieties.

A method of detecting a first target analyte and a second target analyte in a liquid sample, the method comprising:

• (a) contacting a liquid sample containing or suspected of containing a first target analyte and/or a second target analyte with:
• (i) a plurality of first detection probes that specifically bind to the first target analyte;
• (ii) a plurality of first capture probes, the first capture probes being linked to one or more immobilized first target analytes, wherein the first detection probes competitively bind to the first target analytes contained in the liquid sample and to the immobilized first target analytes;
• (iii) a plurality of second detection probes that specifically bind to the second target analyte;
• and
• (iv) a plurality of second capture probes, the second capture probes being linked to one or more immobilized second target analytes, wherein the second detection probes competitively bind to the second target analytes contained in the liquid sample and to the immobilized second target analytes,
• wherein the first capture probes and the second capture probes are detectably and distinguishably labeled;
• (b) incubating the product of step (a) to allow binding of (i) the first detection probes to the first target analytes contained in the liquid sample or to the immobilized first target analytes, and (ii) the second detection probes to the second target analytes contained in the liquid sample or to the immobilized second target analytes;
• (c) separating at least a portion of the first and the second capture probes from the liquid sample;
• (d) labeling with detectable moieties (i) the first detection probes bound to the immobilized first target analytes linked to the first capture probes of step (c), and (ii) the second detection probes bound to the immobilized second target analytes linked to the second capture probes of step (c);
• and
• (e) detecting the first capture probes of step (d), the second capture probes of step (d), and the detectable moieties associated with the first and second capture probes of step (d), thereby detecting the first target analyte and the second target analyte in the liquid sample, wherein the concentration of the first target analyte in the liquid sample is inversely proportional to a signal of the detectable moieties associated with the first capture probes of step (d), and the concentration of the second target analyte in the liquid sample is inversely proportional to a signal of the detectable moieties associated with the second capture probes of step (d).

A method of detecting a first target analyte and a second target analyte in a liquid sample, wherein the first target analyte is a small molecule and the second target analyte is a polypeptide, the method comprising:

• (a) contacting a liquid sample containing or suspected of containing a first target analyte and/or a second target analyte with:
• (i) a plurality of first detection probes that specifically bind to the first target analyte;
• (ii) a plurality of first capture probes, the first capture probes being linked to one or more immobilized first target analytes, wherein the first detection probes competitively bind to the first target analytes contained in the liquid sample and to the immobilized first target analytes;
• (iii) a plurality of second detection probes that specifically bind to the second target analyte;
• and
• (iv) a plurality of second capture probes, the second capture probes being linked to one or more capture ligands, wherein the capture ligand specifically binds to the second target analyte,
• wherein the first capture probes and the second capture probes are detectably and distinguishably labeled;
• (b) incubating the product of step (a) to allow binding of (i) the first detection probes to the first target analytes contained in the liquid sample or to the immobilized first target analytes, and (ii) the second target analytes contained in the liquid sample to the second detection probes and to the capture ligands;
• (c) separating at least a portion of the first and second capture probes from the liquid sample;
• (d) labeling with detectable moieties (i) the first detection probes bound to the immobilized first target analytes linked to the first capture probes of step (c), and (ii) the second detection probes bound to second target analytes, the second target analytes bound to the capture ligands linked to the second capture probes of step (c);
• and
• (e) detecting the first capture probes of step (d), the second capture probes of step (d), and the detectable moieties associated with first and second capture probes of step (d), thereby detecting the first target analyte and the second target analyte in the liquid sample, wherein the concentration of the first target analyte in the liquid sample is inversely proportional to a signal of the detectable moieties associated with the first capture probes of step (d), and the concentration of the second target analyte in the liquid sample is proportional to a signal of the detectable moieties associated with the second capture probes of step (d).

A composition comprising:

• (a) a paramagnetic bead, the paramagnetic bead being linked to one or more immobilized target analytes, wherein the immobilized target analyte is a small molecule;
• (b) an antibody, the antibody being linked to a biotin moiety;
• and
• (c) a beta-galactosidase enzyme, the beta-galactosidase enzyme being linked to a streptavidin moiety,
• wherein the antibody is bound to one of the immobilized target analytes, and the beta-galactosidase enzyme is bound to the antibody by binding of the biotin moiety to the streptavidin moiety.

A composition comprising:

• (a) a paramagnetic bead, the paramagnetic bead being linked to one or more capture antibodies;
• (b) a detection antibody;
• and
• (c) a beta-galactosidase enzyme linked to an immobilized target ligand,
• wherein the detection antibody is bound by one of the capture antibodies, and the detection antibody is bound to the immobilized target ligand.