Electrochemiluminescent labeled probes for use in immunoassay methods, methods using such and kits comprising same

A method of measuring electrochemiluminescence comprising:

• (a) applying a potential to an electrode under conditions in which a complex that is in proximity to the electrode will emit electrochemiluminescence, wherein the complex comprises a target oligonucleotide and a labeled probe of Formula I or Formula II:
• wherein B is a nucleotide base, R is an electrochemiluminescent label, L1 is a linking group, L2 is a linking group, j is an integer between 0 and 11, k is an integer between 0 and 1, m is an integer between 0 and 11, and n is an integer between 0 and 5;
• wherein j is an integer between 0 and 11, k is an integer between 0 and 1, m is an integer between 0 and 11, n is independently an integer between 0 and 5, and R is an electrochemiluminescence label:
• wherein the oligonucleotide of the labeled probe of Formula I or Formula II is complementary to the target oligonucleotide;
• and
• (b) measuring the emitted electrochemiluminescence.

A oligonucleotide-containing compound selected from (I), (II), and (III):

• (I) a non-naturally occurring nucleic acid probe comprising an oligonucleotide, wherein the oligonucleotide is 14-24 nucleotides in length and comprises 14 or 15 contiguous nucleotides of 5′-GACAGAACTAGACAC-3′ (SEQ ID NO:33);
• (II) a non-naturally occurring oligonucleotide comprising 53 to 76 nucleotides, comprising at its 5′ end sequence 5′-GTTCTGTC-3′ and at its 3′ end sequence 5′-GTGTCTA-3′;
• (III) anchoring reagent of Formula X:
• wherein the oligonucleotide of Formula X comprises
• (SEQ ID NO:
• 35)
• 5'-AAGAGAGTAGTACAGCA-3'

A kit for conjugating a nucleic acid probe to a non-nucleic acid detection reagent to form a conjugate, comprising:

• (a) a heterobifunctional cross-linking agent comprising
• (i) a first reactive group capable of reacting with the detection reagent to attach the cross-linking agent to the detection reagent;
• and
• (ii) a second reactive group capable of reacting with the nucleic acid probe to attach the cross-linking agent to the nucleic acid probe, while being substantially unreactive to the detection reagent;
• (b) a first size separation device capable of separating the conjugate from unreacted nucleic acid probe;
• and
• (c) a nucleic acid binding fluorophore, wherein the fluorophore's fluorescence intensity increases when the fluorophore is bound to nucleic acid.

A method of conjugating a nucleic acid probe to a non-nucleic acid detection reagent to form a conjugate, wherein the method comprises (I), (II), or (III):

• (I):
• (a) reacting a detection reagent and a nucleic acid probe to form a conjugate;
• (b) using a size separation device to separate the conjugate from unreacted nucleic acid probe to form purified conjugate;
• (c) forming a test composition comprising a sample of the purified conjugate and a nucleic acid binding fluorophore selected for having a fluorescence intensity that increases when the fluorophore bound to nucleic acid;
• and
• (d) measuring the fluorescence of the test composition to determine an amount of nucleic acid probe in the purified conjugate;
• (II):
• contacting the detection reagent and the nucleic acid probe with a heterobifunctional cross-linking agent under conditions where the detection reagent reacts with a first reactive group of the cross-linking agent and the nucleic acid reacts with a second reactive group of the cross-linking agent to form the conjugate, wherein the heterobifunctional cross-linking agent comprises:
• (i) a first reactive group capable of reacting with the detection reagent to attach the cross-linking agent to the detection reagent and
• (ii) a second reactive group capable of reacting with the nucleic acid probe to attach the cross-linking agent to the nucleic acid probe, while being substantially unreactive to the detection reagent,
• wherein the method does not comprise purifying a reaction product of the detection reagent and the cross-linking agent, prior to the reaction of the cross-linking agent with the nucleic acid probe;
• (III):
• (a) contacting the detection reagent with a heterobifunctional cross-linking agent under conditions where the detection reagent reacts with a first reactive group of the cross-linking agent to form a first composition, wherein the heterobifunctional cross-linking agent comprises
• (i) a first reactive group capable of reacting with the detection reagent to attach the cross-linking agent to the detection reagent and
• (ii) a second reactive group capable of reacting with the nucleic acid probe to attach the cross-linking agent to the nucleic acid probe, while being substantially unreactive to the detection reagent;
• (b) contacting the first composition with the nucleic acid probe under conditions where the second reactive group in the cross-linking agent reacts with the nucleic acid probe to form the conjugate,
• wherein the method does not comprise purifying the reaction product of the detection reagent and the cross-linking agent, prior to the reaction of the cross-linking agent with the nucleic acid probe.

A method of measuring an analyte comprising:

• (a) binding the analyte to (i) a capture reagent in a binding domain on a surface, wherein the binding domain further comprises an anchoring reagent comprising an anchoring oligonucleotide and (ii) a conjugate comprising a detection reagent and a nucleic acid probe;
• thereby forming a complex in the binding domain comprising the capture reagent, the analyte and the conjugate;
• (b) extending the nucleic acid probe of the conjugate to form an extended sequence comprising an anchoring oligonucleotide complement that is complementary to the anchoring oligonucleotide and a detection sequence complement that is complementary to a detection oligonucleotide of a labeled probe;
• (c) binding the labeled probe comprising the detection oligonucleotide to the extended sequence;
• and
• (d) measuring the amount of labeled probe bound to the binding domain by performing the method of claim 1.