Process for complex binding of targeted molecular species

Claim 3

An assay to determine the presence or concentration of at least one selected substance in a fluid comprising: (a) conjugating a first label to a first moiety and a second label to a second moiety, said first and second moieties complimentarily binding to complimentary entities; and (b) detecting a signal indicating the presence or concentration of said substance, said signal being produced in the presence of a first substrate, by an interaction between said first label and said second label, said signal being enhanced by the close proximity of said labels.

Claim 4

An assay to determine the presence or concentration of at least one selected substance in a fluid comprising: (a) conjugating a first label to a first moiety and a second label to a second moiety, said first and second moieties complimentarily binding to complimentary entities; and (b) detecting a signal indicating the presence or concentration of said substance, said signal being produced in the presence of a first substrate, by an interaction between said first label and said second label, said signal being enhanced by the close proximity of said labels.

Claim 5

A nucleotide assay to determine a sequence of nucleotides in a fluid, comprising: (a) binding a target linear sequence of DNA or RNA nucleotides in a single strand to a solid phase; (b) conjugating a first label to a first sequence of nucleotides complementary to said first portion of said target sequence and conjugating a second label to a second sequence of nucleotides complementary to said second portion of said target sequence, said first and second sequences of nucleotides binding to said first and second portions of said target sequence; and (c) detecting a signal produced in the presence of a first substrate, by an interaction between said first label and said second label, enhanced by the proximity to each other by said first and second portions of said target nucleotide sequence.

Claim 6

A nucleotide assay to determine a sequence of nucleotides in a fluid, comprising: (a) binding a target linear sequence of DNA or RNA nucleotides in a single strand to a solid phase; (b) conjugating a first label to a first sequence of nucleotides complementary to said first portion of said target sequence and conjugating a second label to a second sequence of nucleotides complementary to said second portion of said target sequence, said first and second sequences of nucleotides binding to said first and second portions of said target sequence; and (c) detecting a signal produced in the presence of a first substrate, by an interaction between said first label and said second label, enhanced by the proximity to each other by said first and second portions of said target nucleotide sequence.

Claim 7

The nucleotide assay according to claim 3 wherein said target nucleotide sequence is bound to a solid phase.

Claim 8

The nucleotide assay according to claim 3, wherein said first label and said second label are each enzymes, and said signal is produced by the sequential action of said first enzyme label on or in the presence of a first substance, and said second enzyme label on or in the presence of a second substrate, said second substrate being a product of the action of said first enzyme label on said first substrate.

Claim 9

The nucleotide assay according to claim 5, wherein said signal is an increased rate of disappearance of said first substrate.

Claim 10

The nucleotide assay according to claim 5, wherein said signal is an increased rate of disappearance of said second substrate.

Claim 11

The nucleotide assay according to claim 5, wherein said signal is an increased rate of a set of cyclic reactions, in which said first substrate is regenerated by the action of said second enzyme label on or in the presence of said second substrate.

Claim 12

The nucleotide assay according to claim 5, further including the step of adding a scavenger to said fluid to scavenge any second substrate produced by any unconjugated first label in the fluid by the action of said scavenger, to prevent its reaction with the second label.

Claim 13

A process for determining the presence or concentration of a nucleotide sequence in a fluid, comprising the steps of: (a) contacting a solution containing a target linear sequence of DNA or RNA nucleotides in a single strand, having first and second portions, with a solution containing a first sequence of nucleotides conjugated to a first label and complimentary to said first portion of said target sequence, and a second sequence of nucleotides conjugated to a second label and complimentary to said second portion of said target sequence, said first and second complimentary sequences of nucleotides binding to said first and second portions of said target sequence; (b) contacting the fluid with a substrate for said first label, in order to form a first reaction product from said first label and said substrate; (c) measuring a signal produced by the reaction of said second label in the presence of said first reaction product; (d) (i) relating the signal measured in step (c) to the signal measured for a control sample prepared according to steps (a)-(c), said control sample being free of target nucleotide sequences, in order to determine the presence of the target nucleotide sequence in said fluid; or (ii) relating the signal measured in step (c) to the signal measured for samples containing known amounts of said target nucleotide sequence prepared according to steps (a)-(c), in order to determine the concentration of said target nucleotide sequence in said fluid.

Claim 14

A process for determining the presence or concentration of a nucleotide sequence in a fluid, comprising the steps of: (a) contacting a solution containing a target linear sequence of DNA or RNA nucleotides in a single strand, having first and second portions, with a solution containing a first sequence of nucleotides conjugated to a first label and complimentary to said first portion of said target sequence, and a second sequence of nucleotides conjugated to a second label and complimentary to said second portion of said target sequence, said first and second complimentary sequences of nucleotides binding to said first and second portions of said target sequence; (b) contacting the fluid with a substrate for said first label, in order to form a first reaction product from said first label and said substrate; (c) measuring a signal produced by the reaction of said second label in the presence of said first reaction product; (d) (i) relating the signal measured in step (c) to the signal measured for a control sample prepared according to steps (a)-(c), said control sample being free of target nucleotide sequences, in order to determine the presence of the target nucleotide sequence in said fluid; or (ii) relating the signal measured in step (c) to the signal measured for samples containing known amounts of said target nucleotide sequence prepared according to steps (a)-(c), in order to determine the concentration of said target nucleotide sequence in said fluid.

Claim 15

A nucleotide assay to determine the presence or concentration of a nucleotide sequence in a fluid, comprising: (a) binding a probe of a DNA or RNA nucleotide sequence with a complimentary nucleotide sequence; (b) destroying unbound probe and unbound target nucleotide sequences by action of the enzyme nuclease S1, the bound portions of said probe sequence and complimentary sequence being uneffected by the action of said nuclease, said nuclease subsequently being inactivated; (c) adding a homopolymer of Thymidine triphosphate to the 3 ′OH end of said probe and target nucleotide sequences by action of a terminal transferase enzyme; (d) conjugating one set of short sequences of poly Adenosine with a first label, and a second set of said poly Adenosine to a second label, said first and second sets of labeled poly Adenosine binding to said homopolymers of Thymidine triphosphate; and (e) detecting a signal produced in the presence of a first substrate, by an interaction between said first label and said second label, enhanced by their proximity to each other by said bound sequences of poly Adenosine.

Claim 16

A nucleotide assay to determine the presence or concentration of a nucleotide sequence in a fluid, comprising: (a) binding a probe of a DNA or RNA nucleotide sequence with a complimentary nucleotide sequence; (b) destroying unbound probe and unbound target nucleotide sequences by action of the enzyme nuclease S1, the bound portions of said probe sequence and complimentary sequence being uneffected by the action of said nuclease, said nuclease subsequently being inactivated; (c) adding a homopolymer of Thymidine triphosphate to the 3 ′OH end of said probe and target nucleotide sequences by action of a terminal transferase enzyme; (d) conjugating one set of short sequences of poly Adenosine with a first label, and a second set of said poly Adenosine to a second label, said first and second sets of labeled poly Adenosine binding to said homopolymers of Thymidine triphosphate; and (e) detecting a signal produced in the presence of a first substrate, by an interaction between said first label and said second label, enhanced by their proximity to each other by said bound sequences of poly Adenosine.

Claim 17

The nucleotide assay according to claim 11, wherein said first label and said second label are each enzymes, and said signal is produced by the sequential action of said first enzyme label on or in the presence of a first substrate, and said second enzyme label on or in the presence of a second substrate, said second substrate being a product of the action of said first enzyme label on said first substrate.

Claim 18

The nucleotide assay according to claim 12, wherein said signal is an increased rate of disappearance of said first substrate.

Claim 19

The nucleotide assay according to claim 12, wherein said signal is an increased rate of disappearance of said second substrate.

Claim 20

The nucleotide assay according to claim 12, wherein said signal is an increased rate of a set of cyclic reactions, in which said first substrate is regenerated by the action of said second enzyme label on or in the presence of said second substrate.

Claim 21

The nucleotide assay according to claim 12 further including the step of adding a scavenger to said fluid to scavenge any second substrate produced by any unconjugated first label in the fluid by the action of said scavenger, to prevent its reaction with the second label.

Claim 22

A process for determining the presence or concentration of a nucleotide sequence in a fluid, comprising the steps of: (a) contacting the fluid containing a target nucleotide sequence of RNA and DNA in a single linear strand, with a solution of a probe complimentary nucleotide sequence, binding said target sequence and said probe complimentary sequence; (b) Contacting the fluid with a solution of the enzyme, nuclease S1, destroying unbound target and probe sequences, said bound portions of said probe and target sequences being uneffected by the action of said nuclease S1, with subsequent inactivation of said nuclease S1; (c) contacting the fluid with a solution containing a terminal nucleotide transferase to catalyze the 3 ′-OH addition of a homopolymer of Thymidine triphosphate; (d) contacting the fluid with a solution of first and second sets of short sequences of poly Adenosine, said first set of poly Adenosine conjugating to a set of first label and said second set of poly Adenosine conjugating to a set of second label, said two sets of first and second labeled poly Adenosine binding to said homopolymers of Thymidine triphosphate; (e) contacting the fluid with a solution of a substrate for said first label, in order to form a first reaction product from said first label and said substrate; (f) measuring a signal produced by the reaction of said second label in the presence of said first reaction product; (g) (i) relating the signal measured in step (f) to the signal measured for a control sample prepared according to steps (a)-(f) said control sample being free of said target nucleotide sequence, in order to determine the presence of the target sequence in said fluid; or (ii) relating the signal measured in step (f) to the signal measured for samples containing known amounts of said target nucleotide sequence prepared according to steps (a)-(f), in order to determine the concentration of the target nucleotide sequence in said fluid.

Claim 23

A process for determining the presence or concentration of a nucleotide sequence in a fluid, comprising the steps of: (a) contacting the fluid containing a target nucleotide sequence of RNA and DNA in a single linear strand, with a solution of a probe complimentary nucleotide sequence, binding said target sequence and said probe complimentary sequence; (b) Contacting the fluid with a solution of the enzyme, nuclease S1, destroying unbound target and probe sequences, said bound portions of said probe and target sequences being uneffected by the action of said nuclease S1, with subsequent inactivation of said nuclease S1; (c) contacting the fluid with a solution containing a terminal nucleotide transferase to catalyze the 3 ′-OH addition of a homopolymer of Thymidine triphosphate; (d) contacting the fluid with a solution of first and second sets of short sequences of poly Adenosine, said first set of poly Adenosine conjugating to a set of first label and said second set of poly Adenosine conjugating to a set of second label, said two sets of first and second labeled poly Adenosine binding to said homopolymers of Thymidine triphosphate; (e) contacting the fluid with a solution of a substrate for said first label, in order to form a first reaction product from said first label and said substrate; (f) measuring a signal produced by the reaction of said second label in the presence of said first reaction product; (g) (i) relating the signal measured in step (f) to the signal measured for a control sample prepared according to steps (a)-(f) said control sample being free of said target nucleotide sequence, in order to determine the presence of the target sequence in said fluid; or (ii) relating the signal measured in step (f) to the signal measured for samples containing known amounts of said target nucleotide sequence prepared according to steps (a)-(f), in order to determine the concentration of the target nucleotide sequence in said fluid.

Claim 24

A toxigenic complex on a cell membrane in a fluid, comprising: (a) binding selected molecular moieties on said cell membrane to two sets of a binding molecule capable of selectably binding to said molecular moiety, said first set of binding molecule being conjugated to a set of first reactive center and said second set of said binding molecule being conjugated to a set of second reactive center; and (b) producing a substance toxic to said cell by an interaction between said first reactive center and said second reactive center, enhanced by their proximity to each other by the density of said molecular moiety on said cell membrane.

Claim 25

A toxigenic complex on a cell membrane in a fluid, comprising: (a) binding selected molecular moieties on said cell membrane to two sets of a binding molecule capable of selectably binding to said molecular moiety, said first set of binding molecule being conjugated to a set of first reactive center and said second set of said binding molecule being conjugated to a set of second reactive center; and (b) producing a substance toxic to said cell by an interaction between said first reactive center and said second reactive center, enhanced by their proximity to each other by the density of said molecular moiety on said cell membrane.

Claim 26

The toxigenic complex according to claim 18 wherein said first reactive center and said second reactive center are each enzymes, and said toxin is produced by the sequential action of said first enzyme reactive center on or in the presence of a first substrate, and said second enzyme reactive center on or in the presence of a second substrate, said second substrate being a product of the action of said first enzyme reactive center on said first substrate.

Claim 27

A process for producing a toxigenic complex on a cell membrane in a fluid, comprising the steps of: (a) contacting a fluid containing said cell, said cell having selected molecular moieties on the membrane, with a solution of two sets of binding molecule capable of selectively binding to said selected molecular moieties, said first set of binding molecule being conjugated to a set of first reactive center and said second set of binding molecule being conjugated to a set of second reactive center, said first and second sets of binding molecule binding to said molecular moieties on said membrane; (b) contacting the fluid with a substrate for said first reactive center, in order to form a first reaction product from said first reactive center and said substrate, said second reactive center in the presence of said first reaction product producing a substance toxic to said cell.

Claim 28

A process for producing a toxigenic complex on a cell membrane in a fluid, comprising the steps of: (a) contacting a fluid containing said cell, said cell having selected molecular moieties on the membrane, with a solution of two sets of binding molecule capable of selectively binding to said selected molecular moieties, said first set of binding molecule being conjugated to a set of first reactive center and said second set of binding molecule being conjugated to a set of second reactive center, said first and second sets of binding molecule binding to said molecular moieties on said membrane; (b) contacting the fluid with a substrate for said first reactive center, in order to form a first reaction product from said first reactive center and said substrate, said second reactive center in the presence of said first reaction product producing a substance toxic to said cell.