Patent ID: 7238789

Claim:
A method of bead-based immunocapture comprising the steps of binding a conjugate to an antibody-coated bead and detecting a fluorescence signal from bound conjugate, wherein the conjugate is defined by the formula: P-L-DYE, wherein P is a polypeptide; L is a linker; and DYE comprises a compound defined by the formula: including any associated counter ions, wherein: R 1 , R 2 , R 4 , R 6 , R 11 , R 12 , R 13 and 14 , when taken alone, are separately hydrogen sulfonate, carboxylate, phosphonate, phosphate, halogen, C 1 –C 6 alkyl, C 1 –C 6 aminoalkyl, C 5 –C 14 axyl, C 5 –C 14 aryl substituted with one or more of the same or different W groups, —OR A , —SR A ,—NR A R B , —CN, —NO 2 , —C(O)R A or L; R 1 when taken together with R 2 is C 5 –C 14 aryleno or C 5 –C 14 aryleno substituted with one or more of the same or different W groups; R 3 R 3′ , R 7 and R 7′ , when taken alone, are separately hydrogen, L, an aliphatic cationic chain, C 1 –C 6 alkyl or C 5 –C 14 aryl; R 3 when taken together with R 3′ is C 2 –C 8 alkyldiyl; R 7 when taken together with R 7′ is C 2 C 8 alkyldiyl; R 11 and R 12 , when taken together, are C 5 –C 14 aryleno or C 5 –C 14 aryleno substituted with one or more of the same or different W groups; R 12 and R 13 , when taken together, are C 5 –C 14 aryleno or C 5 –C 14 aryleno substituted with one or more of the same or different W groups; R 13 and R 14 , when taken together, are C 5 –C 14 aryleno or C 5 –C 14 aryleno substituted with one or more of the same or different W groups; optionally R 2 and R 3 taken together with the C2-ring atom, C3 ring atom, and 3-nitrogen atom, form a 5- to 7-member ring whose ring atoms are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; optionally R 3′ and R 4 taken together with the 3-nitrogen atom, C3-ring atom, and C4-ring atom, form a 5- to 7-member ring whose ring atoms are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; optionally R 6 and R 7′ taken together with the C6-ring atom, C7-ring atom, and 7-nitrogen atom, form a 5- to 7-member ring whose ring atoms are selected from the group consistirnr of carbon, nitrogen, oxygen, and sulfur; optionally R 7 and R 14 taken together with the 7-nitrogen atom, C7-ring atom, C8-ring atom, and C14-ring atom, form a 5- to 7-member ring whose rina atoms are selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; each W is independently hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C 1 –C 6 alkyl, —OR A , —SR A , —NR A R B , —CN, —NO 2 or —C(O)R A ; and each R A and each R B is independently hydrogen or C 1 –C 6 alkyl; with the proviso that at least one of R 1 , R 2 , R 4 , R 6 , R 11 , R 12 , R 13 and R 14 is sulfonate, or R 12 and R 13 taken together are benzo containing at least one sulfonate attached to the beuzo ring, or at least one of 3 , R 3′ R 7 and R 1′ is C 1 –C 6 alkylsulfonate or C 4 –C 10 arylsulfonate.