Water-soluble conjugates and methods of preparation
The present invention provides water-soluble conjugates and methods of using them in diagnostic and detection assays. Devices for performing detection and quantitation assays are also provided. In various embodiments the conjugates are useful in immunoassays and later flow assays. The invention also provides methods of preparing the conjugates that result in higher yields and higher sensitivities for the assays.
1 - 60 . (canceled)
61 . A method for preparing a water-soluble conjugate, comprising:
simultaneously reacting a water-soluble intermediate precursor comprising a carrier and a linker with a targeting element for a ligand to be detected or a ligand to be detected, and a non-specific protein;
forming a suspension containing a precipitate of the water-soluble conjugate; and
extracting the water-soluble conjugate from the suspension.
62 . The method of claim 61 wherein the water-soluble intermediate precursor further comprises a spacer component and a signal component.
63 . The method of claim 62 wherein the non-specific protein is selected from the group consisting of: bovine serum albumin, an immunoglobulin, and keyhole limpet hemocyanin.
64 . The method of claim 62 wherein the water soluble intermediate precursor is contacted with the targeting element for a ligand to be detected or ligand to be detected and non-specific protein in the presence of a lyotropic salt at a concentration of at least about 1.25 M.
65 . The method of claim 64 wherein the lyotropic salt comprises components selected from the group consisting of: a sulphate, a phosphate, a citrate or tartrate of lithium, sodium, potassium, calcium and ammonium.
66 . The method of claim 65 wherein lyotropic salt is present at a concentration of about 2.5 M and the salt is selected from the group consisting of:
potassium phosphate and sodium phosphate.
67 . The method of claim 62 wherein the water-soluble conjugate is extracted by centrifugation.
68 . The method of claim 67 further comprising applying the water-soluble conjugate to a gel filtration column after extraction.
69 . The method of claim 62 wherein the carrier is dextran.
70 . The method of claim 62 wherein the linker is divinyl sulfone.
71 . The method of claim 62 wherein the spacer is selected from the group consisting of: a polypeptide, bovine serum albumin, ovalbumin, and a globulin.
72 . The method of claim 62 wherein the spacer is bovine serum albumin.
73 . The method of claim 62 wherein the signal component is a dye other than a particulate dye.
74 . The method of claim 73 wherein the dye is rhodamine.
75 . The method of claim 62 wherein the signal component comprises a donor dye and an acceptor dye that perform fluorescent resonance energy transfer (FRET) upon excitation.
76 . The method of claim 62 wherein
the spacer and the non-specific protein are independently and covalently attached to the carrier via the linker;
the signal component is a dye covalently attached to the spacer;
the ligand or targeting element for a ligand is covalently attached to the carrier.
77 . The method of claim 76 further comprising that
the carrier is dextran;
the linker is divinylsulfone; and
the spacer is bovine serum albumin.
78 . The method of claim 62 wherein the targeting element for a ligand is selected from the group consisting of: a monoclonal or polyclonal antibody, a receptor molecule, a lectin, avidin, streptavidin, and biotin.
79 . The method of claim 78 wherein the ligand is selected from the group consisting of: a bacterial or viral antigen, human chorionic gonadotropin (hCG), luteinizing hormone (LH), and a drug of abuse.