Office Action 4: Notice of Allowance (2025-01-03)

CLAIMS:
AMENDMENTS TO THE CLAIMS

What is claimed is:

1\. (Currently Amended) A live cell flow cytometry method for detecting a target of asample comprising living cells, comprising:i) mixing the sample comprising living cells with a molecular probe and adetectable component, wherein:

1) the molecular probe comprises a binding moiety conjugated to a first oligonucleotide sequence, wherein a. the binding moiety comprises an antibody, an antibody-fragment, an enzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, a chelator, or combinations or derivatives thereof; andb. the conjugation between the binding moiety and the first oligonucleotides comprises one or more covalent bond linkages selected from the group consisting of a hydrazine, an oxime, and a triazine; and

2) the detectable component is not immobilized on a solid support and comprises a signal generator conjugated to a second oligonucleotide having a sequence complementary to the sequence of the first oligonucleotide, wherein a. the signal generator comprises a scaffold molecule further conjugated to more than one signal generating moieties, the scaffold molecule comprising a dendrimer, a polysaccharide, or combinations or derivatives thereof; andb. the second oligonucleotide is directly conjugated to the scaffold molecule of the signal generator via one or more covalent bond

linkages selected from the group consisting of a hydrazine, an oxime, and a triazine;
ii) binding the target in the sample with the binding moiety of the molecular probe;
iii) hybridizing the first oligonucleotide with the second oligonucleotide;
iv) reducing the potential for cross-talk by adding to the molecular probe and the detectable component an oligonucleotide comprising at least one of:

1) an unconjugated oligonucleotide having a sequence complementary to the sequence of the first oligonucleotide; and 2) an unconjugated oligonucleotide having a sequence complementary to the sequence of the second oligonucleotide; then v) detecting a signal generated from the detectable component hybridized to the target- bound molecular probe using flow cytometry, wherein the hybridized detectable component is not immobilized on a solid support; and
vi) removing the hybridized detectable component from the bound target, wherein the removal is by a stripping and washing process.
2\. (Previously presented) The method of claim 1, wherein:the molecular probe and the detectable component are combined prior to adding the detectable component to the combined molecular probe and sample; or the molecular probe and the sample are combined prior to adding the detectable component to the combined molecular probe and sample; or the detectable component and the sample are combined prior to adding the molecular probe to the combined detectable component and sample.
3\. (Previously presented) The method of claim 1, wherein:

the molecular probe is bound to the target in the sample prior to hybridizing the first oligonucleotide with the second oligonucleotide; or the first oligonucleotide is hybridized with the second oligonucleotide prior to binding the target in the sample.
4\. (Canceled)
5\. (Canceled)
6\. (Canceled)
7\. (Previously presented) The method of claim 1, wherein:
the detecting comprises a singleplex assay or multiplex assay; and
the detecting further detects, measures, or quantifies the level of binding or amount of the target present in the sample by employing one or more techniques selected from the group consisting of:
chemiluminescence, infrared, microscopy, imaging, high content screening, mass cytometry, lateral flow immunoassay, immunodetection, immunoturbidity, latex agglutination, gold particle agglutination, visual inspection, a change in light transmittance through the sample, increased light transmittance through the sample, immunohistochemistry, immunocytochemistry, in situ hybridization, enzyme immuno-assay, enzyme linked immuno-assay, ELISpot, a blotting method, a Western blot, a Southern blot, a Southwestern blot, labeling inside an electrophoresis system, labeling on a surface, labeling on an array, PCR amplification, elongation followed by PCR amplification, immunoprecipitation, co- immunoprecipitation, chromatin immunoprecipitation, pretargeting imaging, therapeutic agent, and combinations thereof.
8\. (Previously presented) The method of claim 1, wherein the prepared molecular probe and prepared detectable component have at least 90% purity.

9\. (Previously presented) The method of claim 1, further comprising preparing and isolating the molecular probe, wherein the preparing and isolating comprise:i) providing the binding moiety;
ii) conjugating the binding moiety with the first oligonucleotide at greater than 90% efficiency to form a conjugation mixture comprising a binding moiety- oligonucleotide conjugate; andiii) isolating the binding moiety-oligonucleotide conjugate from the conjugation mixture by at least one of binding, retaining, or retarding a substantial portion of:1) the binding moiety-oligonucleotide conjugate, then removing by washing a substantial portion of unconjugated first oligonucleotide, followed by releasing bound, retained, or retarded conjugate; or 2) the unconjugated first oligonucleotide, followed by collecting a substantial portion of at least one of non-bound, non-retained, or non-retarded conjugate by washing.

10\. (Previously presented) The method of claim 9, wherein the isolating utilizes a step selected from the group consisting of chromatography, affinity chromatography, size exclusion chromatography, HPLC, reverse-phase chromatography, electrophoresis,capillary electrophoresis, polyacrylamide gel electrophoresis, agarose gel electrophoresis,
free flow electrophoresis, differential centrifugation, thin layer chromatography,
immunoprecipitation, hybridization, solvent extraction, dialysis, filtration, diafiltration,
tangential flow filtration, ion exchange chromatography, hydrophobic interaction chromatography, and combinations thereof.
11\. (Previously presented) The method of claim 1, further comprising preparing and isolating the detectable component, wherein the preparing and isolating comprises:i) providing a plurality of the signal generators;

ii) conjugating the second oligonucleotide with at least one of the plurality of the signal generator at greater than 90% efficiency to form a conjugation mixture comprising a signal generator-second oligonucleotide conjugate; and
iii) isolating the signal generator-second oligonucleotide conjugates from the conjugation mixture by at least one of binding, retaining, or retarded a substantial portion of:1) the signal generator-second oligonucleotide conjugate, then removing a substantial portion of the unconjugated second oligonucleotide by washing, followed by releasing the bound, retained, or retarded conjugate;
or 2) unconjugated second oligonucleotide, followed by collecting a substantial portion of at least one of the non-bound, non-retained, or non-retarded conjugate by washing.

12\. (Canceled)

13\. (Previously presented) The method of claim 1, wherein the scaffold molecule further comprises a component selected from the group consisting of a dextran, a protein, a peptide, a further oligonucleotide sequence, a portion of the second oligonucleotide that is not complementary to the first oligonucleotide, a polymer, a hydrophilic polymer, andcombinations or derivatives thereof.

14\. (Previously presented) The method of claim 1, wherein the antibody comprises a monoclonal antibody or a polyclonal antibody.

15\. (Original) The method of claim 1, wherein the target is a biological target.

16\. (Previously presented) The method of claim 15, wherein the biological target comprises a component selected from the group consisting of an antigen, a pathogen, a protein, a peptide, an epitope, a carbohydrate-containing molecule, a small molecule, and a combination or derivative thereof.

17\. (Previously presented) The method of claim 11, wherein the plurality of signal generators of the detectable component or a hybridized detectable component comprises one or more components selected from the group consisting of:a directly detectable signal generating moiety, an indirectly detectable signal generating moiety, a fluorescent dye, a fluorophore, a fluorochrome, a chromophore, a biofluorescent protein, a luminescent species, a chemiluminescent compound, a electrochemiluminescent label, a bioluminescent label, a phosphorescent species, a fluorophore labeled DNA dendrimer, Quantum Dot, a tandem dye, a FRET dye, a spin label, a radioactive isotope, a stable isotope, a heavy metal chelate, an enzyme, an enzyme substrate, a molecule specifically recognized by another substance carrying a label or reacts with a substance carrying a label, an antibody, an antibody fragment, an antigen, a nucleic acid, a nucleic acid analog, an oligonucleotide, an oligonucleotide analog, complementary oligonucleotide, a complementary oligonucleotide analog, a ligand, a protein, a peptide ligand, a protein substrate, a receptor, a substrate, a secondary reporter, a hapten, and-combinations or derivatives thereof.

18\. (Previously presented) The method of claim 1, wherein the method comprises an automated system or robotic system that performs one or more steps of the claimed method.

19\. (Canceled)

20\. (Canceled)

21\. (Previously presented) The method of claim 15, wherein the binding moiety is a monoclonal antibody or a polyclonal antibody.

22\. (Previously presented) The method of claim 15, wherein the biological target is a protein or a peptide.

23\. (Previously presented) The method of claim 1, wherein

the first oligonucleotide is a 10-mer to 60-mer oligonucleotide; and
the second oligonucleotide is a 10-mer to 60-mer oligonucleotide.

24\. (Previously presented) The method of claim 1, wherein the molecular probe and the detectable component are combined prior to mixing the sample with the molecular probe and the detectable component.

25\. (Previously presented) The method of claim 24, wherein the first oligonucleotide is hybridized with the second oligonucleotide prior to mixing the sample with the molecular probe and the detectable component.

26\. (Previously presented) The method of claim 1, wherein reducing cross-talk according to step (iv) comprises adding the non-conjugated oligonucleotide(s) to a mixture of the molecular probe and the detectable component before mixing the sample with the molecular probe and the detectable component.

27\. (Previously presented) The method of claim 26, wherein the first oligonucleotide is hybridized with the second oligonucleotide before adding the non-conjugated oligonucleotide(s).

28\. (Previously presented) The method of claim 1, wherein a plurality of targets in the sample are simultaneously detected using a plurality of molecular probe and detectable components, wherein each molecular probe and detectable component pair is hybridized in accord with step (iii) and treated to reduce cross-talk in accord with step (iv) prior to mixing with the sample.

29\. (Previously presented) The method of claim 1, wherein the scaffold molecule comprises dextran.

30\. (Previously presented) The method of claim 1, wherein the scaffold molecule consists essentially of dextran.

31\. (Previously presented) The method of claim 1, wherein the detectable component consists essentially of the second oligonucleotide and a dextran conjugated to

EMDM 0002 (3515781.000201) multiple fluorophores and wherein the second oligonucleotide is directly conjugated to the dextran via a hydrazone, triazine or oxime linkage.

OFFICE ACTION:
Allowance notice
1.	The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination under 37 CFR 1.114
2.	A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.  Applicant's submission filed on 07/03/2024 has been entered.
3.	This application is a CON of 13/302,877 filed on 11/22/2011 now ABN
which claims benefit of 61/483,186 filed on 05/06/2011.
Claim status
4.	In the claim listing filed on 07/03/2024 claims 1-3, 7-11, 13-18 and 21-31 are pending in this application and are under prosecution.
Claim 1 is amended. Claims 4-6, 12 and 19-20 are canceled. The amendments have been reviewed and entered. No new matter has been introduced as applicant has identified the support for the amendments in the instant application.
Withdrawn Rejections and response to the Remarks
5.	The previous rejection of claims 1-3, 8-11, 13-18, and 21-30 under 35 USC 103 (a) as being unpatentable over Winther in view of Schwartz and further in view of Coull has been withdrawn in view of persuasive arguments made by the applicant that Winther only refers to flow cytometry generally. Winther does not teach or suggest a method of live cell flow cytometry or techniques or methodologies that would be adaptable to live cell flow cytometry and further argues that the techniques of Winther are incompatible with keeping living cells in a sample alive during target detection as required by the live cell flow cytometry method of the instant claims. Winther teaches fixatives that are used to preserve cells and tissues in a reproducible and life-like manner and to stabilize cells and tissues to protect them from the rigors of processing and staining techniques (paragraph 0098). None of the working examples of Winther utilize living cells or techniques that could be applied to living cells with any expectation of success (Remarks, pgs. 10).
Applicant further argues that claim 1 as amended and Winther, Schwartz, and Coull and respectfully submit that Salazar fails to overcome these defects. While Salazar is directed to a flow cytometry apparatus that can also perform microscopy, there would have been no motivation to combine Salazar with Schwartz and Coull since Schwartz and Coull are silent on flow cytometry. Furthermore, while Salazar refers to using fixed and unfixed cells (see paragraph 0044), one skilled in the art would not have been motivated to use unfixed cells as required for live cell flow cytometry given the advantages of using fixed cells as taught by Winther (see paragraph [0098]).
With regard to claims 29-31 applicant further argues that the office cites Rowley as teaching methods wherein dextran is linked to fluorescent labels using hydrazine linkages and that the method has the added advantage of allowing direct labeling with multiple labeling entities while overcoming the problem of fluorescence quenching.
Applicants maintain the arguments above concerning claim 1 as amended and Winther, Schwartz, and Coull and argues that Rowley fails to overcome these defects, as it is silent on flow cytometry generally and live cell flow cytometry in particular.
Double patenting rejection withdrawn
6.	With regard to double patenting rejection on the record applicant argues that Claims 1-20 of U.S. Patent No. 9,222,936 B2 do not recite a method including
removing a hybridized detectable component from the bound target, wherein the removal is by a stripping and washing process as called for in instant claim 1. Claims 1-20 of U.S. Patent No. 9,222,936 B2 are also silent on live cell flow cytometry as called for in claim 1 as amended.
Further, in view of the arguments set forth above with respect to the
rejections under 35 U.S.C §103, Applicants respectfully submit that the amended claims
are patentably distinct from the invention defined in the claims of U.S. Patent No.
9,222,936 B2 alone or in combination with Winther, Schwartz, Coull, Salazar, and/or
Rowley.
The arguments are persuasive. Therefore, pending ODP rejection has been withdrawn.
Examiner’s comment
7.	Claims 1-3, 7-11, 13-18 and 21-31 have been renumbered. The dependent claims are grouped together based on their dependencies.
Conclusion
8.	Claims 1-3, 7-11, 13-18 and 21-31 are allowed.

APPLICANT REBUTTAL: REMARKS
Claim 1 has been amended. Claims 1-3, 7-11, 13-18, and 21-31 will be pending upon entry of this amendment.
Support for the amendments can be found in at least paragraphs [0063]-[0066], [0070], and [0241] of the published application, US 2022/0170073 Al.
No new matter is being added with these amendments.
Rej
ections Under 35 U.S.C
1. Winther, Schwartz, and Coull
Reconsideration is respectfully requested of the rejection of claims 1-3, 8-11, 13- 18, and 21-30 under 35 U.S.C. 103(a) as being unpatentable over Winther (WO 2007/031874 A2), Schwartz (US 2003/0013857 Al), and Coull (US 2003/0124521).
Claim 1 as amended recites a live cell flow cytometry method for detecting a target, comprising, inter alia, mixing a sample comprising living cells with a molecular probe and a detectable component.
Winther only refers to flow cytometry generally. None of the working examples are for flow cytometry, and flow cytometry is only mentioned in the specification as part of a list of several different detection systems listed by name in the abstract and at paragraphs [0002] and [0090].
More importantly, Winther does not teach or suggest a method of live cell flow cytometry or techniques or methodologies that would be adaptable to live cell flow cytometry. Winther references that samples may be derived from living matter or a living organism (paragraph [0086]). However, the techniques of Winther are incompatible with keeping living cells in a sample alive during target detection as required by the live cell flow cytometry method of the instant claims. Winther teaches fixatives that are used to preserve cells and tissues in a reproducible and life-like manner and to stabilize cells and tissues to protect them from the rigors of processing and staining techniques (paragraph [0098]). This would necessarily result in cell death.
None of the working examples of Winther utilize living cells or techniques that could be applied to living cells with any expectation of success. Example 22-36 refer to one or more of fixation of biological samples, sample dehydration and paraffin embedding,
and cutting, mounting, and deparaffination of embedded samples. Examples 39-50 all use tissue sections. Each of these methods require killing the cells. The remaining examples all refer to general methods, exemplary conjugates or monomers, or methods of synthesizing them.
Because Schwartz and Coull are silent on flow cytometry, there would have been no motivation to combine Schwartz and Coull with Winther to produce a live cell flow cytometry method as required by the instant claims. Further, nothing in the disclosure of Schwartz and Coull directs or motivates the skilled person to modify the techniques taught in the primary reference so as to render the methods taught therein suitable for live cell flow cytometry.
Accordingly, Applicants respectfully request withdrawal of this rejection.
2. Winther, Schwartz, and Coull in view of Salazar
Reconsideration is respectfully requested of the rejection of claim 7 as under 35 U.S.C. 103(a) as being unpatentable over Winther (WO 2007/031874 A2), Winther (US 2010/0105145 Al), Schwartz (US 2003/0013857 Al), and Coull (US 2003/0124521) in further view of Salazar (US 2009/0051912 Al).
The Office admits that neither Winther, Schwartz, nor Coull explicitly teach the combination of flow cytometry and microscopy. The Office adds that Salazar teaches methods wherein flow cytometry is combined with microscopy, which has the added advantage of allowing analysis of the sizes and attributes of cells without having to replace major components of a flow cytometer. The Office asserts that it would have been obvious to a person of ordinary skill in the art to have combined the method of Winther, Schwartz, and Coull with the teachings of Salazar to arrive at the instantly claimed method with a reasonable expectation of success.
Applicants maintain the arguments above concerning claim 1 as amended and
Winther, Schwartz, and Coull and respectfully submit that Salazar fails to overcome these defects. While Salazar is directed to a flow cytometry apparatus that can also perform microscopy, there would have been no motivation to combine Salazar with Schwartz and Coull since Schwartz and Coull are silent on flow cytometry. Furthermore, while Salazar refers to using fixed and unfixed cells (see paragraph [0044]), one skilled in the art would
not have been motivated to use unfixed cells as required for live cell flow cytometry given the advantages of using fixed cells as taught by Winther (see paragraph [0098]) and detailed above.
Accordingly, Applicants respectfully request withdrawal of this rejection.
3. Winther, Schwartz, and Coull in view of Rowley
Reconsideration is respectfully requested of the rejection of claims 29-31 under 35 U.S.C. 103(a) as being unpatentable over Winther (WO 2007/031874 A2), Winther (US 2010/0105145 Al), Schwartz (US 2003/0013857 Al), and Coull (US 2003/0124521) as applied to claim 1 above and in further view of Rowley (US 4,975,532).
The Office cites Rowley as teaching methods wherein dextran is linked to
fluorescent labels using hydrazine linkages and that the method has the added advantage of allowing direct labeling with multiple labeling entities while overcoming the problem of fluorescence quenching. The Office argues that it would have been obvious to a person of ordinary skill in the art to have combined the method of Winther, Schwartz, and Coull with the teachings of Rowley to arrive at the instantly claimed method with a reasonable expectation of success.
Applicants maintain the arguments above concerning claim 1 as amended and
Winther, Schwartz, and Coull and argues that Rowley fails to overcome these defects, as it is silent on flow cytometry generally and live cell flow cytometry in particular.
Accordingly, Applicants respectfully request withdrawal of this rejection.
Double Patenting
Claims 1-3, 8-11, 13-19, and 21-30 are rejected on the ground of nonstatutory
double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 9,222,936 B2
in view of Winther, Schwartz, and Coull.
Claim 7 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 9,222,936 B2 in view of Winther, Schwartz, and Coull and further in view of Salazar.
Claims 29-31 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 9,222,936 B2 in view of Winther, Schwartz, and Coull and further in view of Rowley.
Claims 1-20 of U.S. Patent No. 9,222,936 B2 do not recite a method including
removing a hybridized detectable component from the bound target, wherein the removal is by a stripping and washing process as called for in instant claim 1. Claims 1-20 of U.S. Patent No. 9,222,936 B2 are also silent on live cell flow cytometry as called for in claim 1 as amended. Further, in view of the arguments set forth above with respect to the rejections under 35 U.S.C § 103, Applicants respectfully submit that the amended claims are patentably distinct from the invention defined in the claims of U.S. Patent No. 9,222,936 B2 alone or in combination with Winther, Schwartz, Coull, Salazar, and/or Rowley.
Accordingly, Applicants respectfully request withdrawal of these rejections.
CONCLUSION


CITED REFERENCE SUMMARIES: Ref US9222936B2: Claims:
What is claimed is: 1. A method for suppressing cross-hybridization in assaying one or more targets of a sample comprising i) hybridizing 1) a molecular probe, comprising a binding moiety conjugated to an oligonucleotide; and 2) a detectable component, comprising a signal generating moiety conjugated to an oligonucleotide having a sequence complementary to the sequence of the oligonucleotide of the molecular probe; ii) blocking the hybridized molecular probe-detectable component with a blocking component comprising 1) an oligonucleotide having a sequence complementary to the sequence of the oligonucleotide of the molecular probe, or 2) an oligonucleotide having a sequence complementary to the sequence of the oligonucleotide of the detectable component; iii) combining the blocked hybridized molecular probe-detectable component with one or more blocked hybridized molecular probe-detectable components similarly prepared by steps i) through ii) to yield a mixture of the blocked hybridized molecular probe-detectable components; iv) combining the mixture of the blocked hybridized molecular probe-detectable components with the sample comprising the one or more targets; v) binding the one or more targets in the sample with one or more of the binding moieties of the molecular probes comprising the mixture of the blocked hybridized molecular probe-detectable components; vi) detecting a signal generated from the one or more targets bound to the one or more of the molecular probes that comprise the mixture of the blocked hybridized molecular probe-detectable components; wherein the method further is characterized by one or more of a) the conjugation between the oligonucleotide and the binding moiety and the conjugation between the complementary oligonucleotide and the signal generating moiety, wherein each conjugation comprises one or more covalent bond linkages comprising a hydrazone, oxime, triazine, or other covalent bond, wherein the formation of the conjugates is at least 90% efficient; and b) the binding moiety comprises a binding affinity for the target of less than 10−4 M. 2. The method of claim 1, wherein the assay comprises a multiplex assay. 3. The method of claim 1, wherein the assay detects, measures, or quantifies at least one of the level of binding or the amount of the target present in the sample by using one or more of flow cytometry, immunomagnetic cellular depletion, immunomagnetic cell capture, array, bead array, multiplex bead array, microarray, antibody array, cellular array, chemiluminescence, infrared, microscopy, imaging, high content screening (HCS), mass cytometry, lateral flow immunoassay, immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot, immunoturbidity, latex agglutination, gold particle agglutination, visual inspection, a change in light transmittance through said sample, increased light transmittance through said sample, a blotting method, a western blot, a Southern blot, a southwestern blot, labeling inside an electrophoresis system, labeling on a surface, labeling on an array, PCR amplification, elongation followed by PCR amplification, immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, pretargeting imaging, therapeutic agent. 4. The method of claim 2, wherein the assay detects, measures, or quantifies at least one of the level of binding or of the amount of the target present in the sample using flow cytometry. 5. The method of claim 1, wherein the sample is characterized as at least one of i) a complex sample or ii) a homogeneous or a heterogeneous mixture; and wherein the sample comprises at least one of a) a range of analytes having a wide range of binding specificities; b) a cell, a membrane, a biological molecule, a metabolite, or a disease biomarker; and c) a biological fluid or a fluidized biological tissue. 6. The method of claim 1, wherein the blocking component comprises the oligonucleotide having a sequence complementary to the sequence of the oligonucleotide of the molecular probe. 7. The method of claim 6, wherein the blocking component is provided in an amount that is about 1 to 200 times greater than the concentration of the oligonucleotide of the molecular probe. 8. The method of claim 6, wherein the blocking component is provided in an amount that is about 10 times greater than the concentration of the oligonucleotide of the molecular probe. 9. The method of claim 6, wherein the blocking component is provided in an amount that is about 25 times greater than the concentration of the oligonucleotide of the molecular probe. 10. The method of claim 6, wherein the blocking component is provided in an amount that is about 50 times greater than the concentration of the oligonucleotide of the molecular probe. 11. The method of claim 1, wherein the blocking component comprises an oligonucleotide having a sequence complementary to the sequence of the oligonucleotide of the detectable component. 12. The method of claim 11, wherein the blocking component is provided in an amount that is about 1 to 200 times greater than the concentration of the oligonucleotide of the molecular probe. 13. The method of claim 11, wherein the blocking component is provided in an amount that is about 10 times greater than the concentration of the oligonucleotide of the molecular probe. 14. The method of claim 11, wherein the blocking component is provided in an amount that is about 25 times greater than the concentration of the oligonucleotide of the molecular probe. 15. The method of claim 11, wherein the blocking component is provided in an amount that is about 50 times greater than the concentration of the oligonucleotide of the molecular probe. 16. The method of claim 1, wherein the method suppresses cross-hybridization about 10% to 100% more than does the method in which the blocking component is omitted. 17. The method of claim 1, wherein the detectable component comprises a scaffold conjugated to the complementary oligonucleotide, wherein the scaffold has one or more signal generating moieties, and wherein the scaffold comprises a dendrimer, a polysaccharide, a dextran, a protein, a peptide, a second oligonucleotide an additional portion of the complementary oligonucleotide that is not complementary to the oligonucleotide of the molecular probe, a polymer, a hydrophilic polymer, a bead, a nanoparticle, or a combination or derivative thereof. 18. The method of claim 1, wherein the binding moiety comprises an antibody, a monoclonal antibody, a polyclonal antibody, an enzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, a small molecule, an aptamer, a chelator, or a combination or derivative thereof. 19. The method of claim 1, wherein the biological target comprises an antigen, a pathogen, a protein, a peptide, an epitope, a carbohydrate-containing molecule, a small molecule, or a combination or derivative thereof. 20. The method of claim 1, wherein the signal generating moiety comprises a directly detectable signal generating moiety, an indirectly detectable signal generating moiety, a fluorescent dye, a fluorophore, a fluorochrome, a chromophore, a fluorescent protein, a biofluorescent protein, a luminescent species, a chemiluminescent compound, a electrochemiluminescent label, a bioluminescent label, a phosphorescent species, a fluorophore labeled DNA dendrimer, a Quantum Dot, a Raman particle, a tandem dye, a FRET dye, a heavy atom, a spin label, a radioactive isotope, a nanoparticle, a light scattering nanoparticle or microsphere, a diffracting particle, a polymer, a polymer particle, a bead, a solid surface, a metal particle, a stable isotope, a heavy metal chelate, a magnetic particle, an RFID tag, a microbarcode particle, an enzyme, an enzyme substrate, a molecule that is specifically recognized by another substance carrying a label or that reacts with another substance carrying a label, an antibody, an antibody fragment, an antigen, a nucleic acid, a nucleic acid analog, an oligonucleotide, an oligonucleotide analog, a complementary oligonucleotide, a complementary oligonucleotide analog, a ligand, a protein, a peptide ligand, a protein substrate, a receptor; a substrate, a secondary reporter, a hapten, or a combination or derivative thereof.
A method for suppressing cross-hybridization in assaying one or more targets of a sample comprising i) hybridizing 1) a molecular probe, comprising a binding moiety conjugated to an oligonucleotide; and 2) a detectable component, comprising a signal generating moiety conjugated to an oligonucleotide having a sequence complementary to the sequence of the oligonucleotide of the molecular probe; ii) blocking the hybridized molecular probe-detectable component with a blocking component comprising 1) an oligonucleotide having a sequence complementary to the sequence of the oligonucleotide of the molecular probe, or 2) an oligonucleotide having a sequence complementary to the sequence of the oligonucleotide of the detectable component; iii) combining the blocked hybridized molecular probe-detectable component with one or more blocked hybridized molecular probe-detectable components similarly prepared by steps i) through ii) to yield a mixture of the blocked hybridized molecular probe-detectable components; iv) combining the mixture of the blocked hybridized molecular probe-detectable components with the sample comprising the one or more targets; v) binding the one or more targets in the sample with one or more of the binding moieties of the molecular probes comprising the mixture of the blocked hybridized molecular probe-detectable components; vi) detecting a signal generated from the one or more targets bound to the one or more of the molecular probes that comprise the mixture of the blocked hybridized molecular probe-detectable components; wherein the method further is characterized by one or more of a) the conjugation between the oligonucleotide and the binding moiety and the conjugation between the complementary oligonucleotide and the signal generating moiety, wherein each conjugation comprises one or more covalent bond linkages comprising a hydrazone, oxime, triazine, or other covalent bond, wherein the formation of the conjugates is at least 90% efficient; and b) the binding moiety comprises a binding affinity for the target of less than 10−4 M.
The method of claim 1, wherein the assay comprises a multiplex assay.
The method of claim 1, wherein the assay detects, measures, or quantifies at least one of the level of binding or the amount of the target present in the sample by using one or more of flow cytometry, immunomagnetic cellular depletion, immunomagnetic cell capture, array, bead array, multiplex bead array, microarray, antibody array, cellular array, chemiluminescence, infrared, microscopy, imaging, high content screening (HCS), mass cytometry, lateral flow immunoassay, immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot, immunoturbidity, latex agglutination, gold particle agglutination, visual inspection, a change in light transmittance through said sample, increased light transmittance through said sample, a blotting method, a western blot, a Southern blot, a southwestern blot, labeling inside an electrophoresis system, labeling on a surface, labeling on an array, PCR amplification, elongation followed by PCR amplification, immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, pretargeting imaging, therapeutic agent.
The method of claim 2, wherein the assay detects, measures, or quantifies at least one of the level of binding or of the amount of the target present in the sample using flow cytometry.
The method of claim 1, wherein the sample is characterized as at least one of i) a complex sample or ii) a homogeneous or a heterogeneous mixture; and wherein the sample comprises at least one of a) a range of analytes having a wide range of binding specificities; b) a cell, a membrane, a biological molecule, a metabolite, or a disease biomarker; and c) a biological fluid or a fluidized biological tissue.
The method of claim 1, wherein the sample is characterized as at least one of i) a complex sample or ii) a homogeneous or a heterogeneous mixture;
and wherein the sample comprises at least one of a) a range of analytes having a wide range of binding specificities; b) a cell, a membrane, a biological molecule, a metabolite, or a disease biomarker; and c) a biological fluid or a fluidized biological tissue.
The method of claim 1, wherein the blocking component comprises the oligonucleotide having a sequence complementary to the sequence of the oligonucleotide of the molecular probe.
The method of claim 6, wherein the blocking component is provided in an amount that is about 1 to 200 times greater than the concentration of the oligonucleotide of the molecular probe.
The method of claim 6, wherein the blocking component is provided in an amount that is about 10 times greater than the concentration of the oligonucleotide of the molecular probe.
The method of claim 6, wherein the blocking component is provided in an amount that is about 25 times greater than the concentration of the oligonucleotide of the molecular probe.
The method of claim 6, wherein the blocking component is provided in an amount that is about 50 times greater than the concentration of the oligonucleotide of the molecular probe.
The method of claim 1, wherein the blocking component comprises an oligonucleotide having a sequence complementary to the sequence of the oligonucleotide of the detectable component.
The method of claim 11, wherein the blocking component is provided in an amount that is about 1 to 200 times greater than the concentration of the oligonucleotide of the molecular probe.
The method of claim 11, wherein the blocking component is provided in an amount that is about 10 times greater than the concentration of the oligonucleotide of the molecular probe.
The method of claim 11, wherein the blocking component is provided in an amount that is about 25 times greater than the concentration of the oligonucleotide of the molecular probe.
The method of claim 11, wherein the blocking component is provided in an amount that is about 50 times greater than the concentration of the oligonucleotide of the molecular probe.
The method of claim 1, wherein the method suppresses cross-hybridization about 10% to 100% more than does the method in which the blocking component is omitted.
The method of claim 1, wherein the detectable component comprises a scaffold conjugated to the complementary oligonucleotide, wherein the scaffold has one or more signal generating moieties, and wherein the scaffold comprises a dendrimer, a polysaccharide, a dextran, a protein, a peptide, a second oligonucleotide an additional portion of the complementary oligonucleotide that is not complementary to the oligonucleotide of the molecular probe, a polymer, a hydrophilic polymer, a bead, a nanoparticle, or a combination or derivative thereof.
The method of claim 1, wherein the binding moiety comprises an antibody, a monoclonal antibody, a polyclonal antibody, an enzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, a small molecule, an aptamer, a chelator, or a combination or derivative thereof.
The method of claim 1, wherein the biological target comprises an antigen, a pathogen, a protein, a peptide, an epitope, a carbohydrate-containing molecule, a small molecule, or a combination or derivative thereof.
The method of claim 1, wherein the signal generating moiety comprises a directly detectable signal generating moiety, an indirectly detectable signal generating moiety, a fluorescent dye, a fluorophore, a fluorochrome, a chromophore, a fluorescent protein, a biofluorescent protein, a luminescent species, a chemiluminescent compound, a electrochemiluminescent label, a bioluminescent label, a phosphorescent species, a fluorophore labeled DNA dendrimer, a Quantum Dot, a Raman particle, a tandem dye, a FRET dye, a heavy atom, a spin label, a radioactive isotope, a nanoparticle, a light scattering nanoparticle or microsphere, a diffracting particle, a polymer, a polymer particle, a bead, a solid surface, a metal particle, a stable isotope, a heavy metal chelate, a magnetic particle, an RFID tag, a microbarcode particle, an enzyme, an enzyme substrate, a molecule that is specifically recognized by another substance carrying a label or that reacts with another substance carrying a label, an antibody, an antibody fragment, an antigen, a nucleic acid, a nucleic acid analog, an oligonucleotide, an oligonucleotide analog, a complementary oligonucleotide, a complementary oligonucleotide analog, a ligand, a protein, a peptide ligand, a protein substrate, a receptor; a substrate, a secondary reporter, a hapten, or a combination or derivative thereof.