Office Action 3: Final Rejection (2024-03-05)
Rejection types: 103 - Obviousness, Double Patenting

CLAIMS:
AMENDMENTS TO THE CLAIMS

What is claimed is:

1\. (Currently Amended) A flow cytometry method for detecting a target of a sample,comprising:i) mixing the sample with a molecular probe and a detectable 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\. (Currently Amended) The method of claim 1, wherein:\[\[the\]\] 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\. (Currently Amended) 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\. (Currently Amended) 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; and
iii) 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\. (Currently Amended) 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\. (Currently Amended) 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:
FINAL ACTION
Notice of Pre-AIA  or AIA  Status
1.	The present application is being examined under the pre-AIA  first to invent provisions.
Status of the Claims
2.	This action is in response to papers filed 19 December 2023 in which claims 1-3, 9, 26, and 28 were amended, claim 19 was canceled, and no new claims were added.  All of the amendments have been thoroughly reviewed and entered.
Any previous rejections not reiterated below are withdrawn in view of the amendments.
Applicant’s arguments have been thoroughly reviewed and are addressed following the rejections necessitated by the amendments.
3.	Claims 1-3, 7-11, 13-19, and 21-31 are under prosecution.
Claim Interpretation
4.	 For the purposes of searching for and applying prior art under 35 U.S.C. 102 and 103, absent a clear indication in the specification or claims of what the basic and novel characteristics actually are, consisting essentially of, as found in claims 30 and 31, will be construed as equivalent to comprising. See, e.g., PPG, 156 F.3d at 1355, 48 USPQ2d at 1355 (PPG could have defined the scope of the phrase consisting essentially of' for purposes of its patent by making clear in its specification what it regarded as constituting a material change in the basic and novel characteristics of the invention.). See also AK Steel Corp. v. Sollac, 344 F3.d 1234, 1239-1240, 68 USPQ2d 1280, 1283-84 (Fed. Cir. 2003); In re Janakirama-Rao, 317 F.2d 951, 954, 137 USPQ 893, 895-96 (CCPA 1963).  See MPEP 2163.
Interview Summary
5.	The interview summary included with the Remarks filed 19 December 2023 is acknowledged and the interview record is complete.
Claim Rejections - 35 USC § 103
6.	The following is a quotation of pre-AIA  35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
7.	This application currently names joint inventors. In considering patentability of the claims under pre-AIA  35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary.  Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA  35 U.S.C. 103(c) and potential pre-AIA  35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA  35 U.S.C. 103(a).
8.	It is noted that the courts have stated “[A] prior art reference must be considered in its entirety, i.e., as a whole” W.L. Gore  & Associates, Inc. v. Garlock, Inc., 721 F.2d 1540, 220 USPQ 303 (Fed. Cir. 1983) (see MPEP 2141.02 VI).  Thus, while the rejections listed below present a modified interpretation of the teachings of the previously cited prior art solely for the purpose of clarity, the claims remain rejected over the prior art of record.
9.	Claims 1-3, 8-11, 13-18, and 21-30 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003).
Regarding claim 1, Winther teaches a method of detecting a target in a sample using flow cytometry (Abstract), comprising providing to the sample a molecular probe, in the form of a recognition unit, having a first oligonucleotide sequence, in the form of a nucleic acid segment, conjugated through a linker to a binding moiety, in the form of an antibody (Figure 3 and paragraph 0013).  Winther further teaches a detectable component, in the form of a detection unit, which comprises a second oligonucleotide (i.e., nucleic acid analog), which hybridizes to the first oligonucleotide sequence via a complementary sequence (e.g., paragraphs 0014 and 0168), and which further comprises a signal generator, in the form of multiple detectable labels, which are attached to a polymer scaffold (Figures 3 and 6, and paragraphs 0013 and 0016).  The polymer comprises a polysaccharide, in the form of the polysaccharide dextran or a dendrimer (paragraphs 0174-0175).
It is noted that the specification states that oligonucleotides comprise nucleobases and analogs thereof (paragraphs 0144-0145); thus, the nucleic acid analogs of Winther are oligonucleotides, and the claim has been given the broadest reasonable interpretation consistent with the teachings of the specification regarding an “oligonucleotide” (In re Hyatt, 211 F.3d1367, 1372, 54 USPQ2d 1664, 1667 (Fed. Cir. 2000) (see MPEP 2111 [R-1]).
Winther also teaches the labels are fluorescent dyes (paragraph 0100); thus, none of the components are immobilized on solid supports.  Winther further teaches the target is detected by binding the probe, and that labeling is later than probe binding (paragraph 0004), thus, it would have been obvious to bind the target to the binding moiety, followed by hybridizing the first and second oligonucleotides by subsequently adding the detection unit.
Alternatively, the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results (In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946).  See MPEP 2144.04 IV.C.
In addition, the courts have found that “where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).  See MPEP 2144.05 II.
Thus, any order of combining the components represents an obvious variant and/or routine optimization of the steps of the prior art.
Applicant is advised that MPEP 716.01(c) makes clear that “[t]he arguments of counsel cannot take the place of evidence in the record” (In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965)). Thus, Applicant should not merely rely upon counsel’s arguments in place of evidence in the record.
It is noted that the Response above should not be construed as an invitation to file an after final declaration.  See MPEP 715.09.
Winther also teaches detection of the signals (i.e., labels) using flow cytometry (Abstract), and that the method has the added advantage of being compatible with many differently types of samples, probes, targets, and labels (Abstract).  Thus, Winther teaches the known techniques discussed above.
While Winther teaches the oligonucleotide within the recognition unit and the oligonucleotide within the detection units are each covalently attached (paragraph 0174), as well as the use of hydrazines (paragraph 0078) and aldehydes (paragraph 0186), which form hydrazones, Winther does not explicitly teach the use of the functionally equivalent hydrazone group.
However, Schwartz teaches a method wherein a biomolecule is attached (i.e., crosslinked) using a hydrazine moiety to link an amino group (Abstract) to form a hydrazine (paragraph 0028).  Schwartz teaches the linking of oligonucleotides and antibodies (paragraph 0016), and that the method has the added advantage of allowing immobilization in a single step (paragraph 0111).  Thus, Schwartz teaches the known techniques discussed above.
It would therefore have been obvious to a person of ordinary skill in the art to have combined the method of Winther with the teachings of Schwartz, which utilizes a functionally equivalent amine derived hydrazine, to arrive at the instantly claimed method with a reasonable expectation of success.  The ordinary artisan would have been motivated to make the combination because said combination would have resulted in a method having the added advantage of being compatible with many differently types of samples, probes, targets, and labels as explicitly taught by Winther (Abstract) as well as the added advantage of allowing immobilization in a single step as explicitly taught by Schwartz (paragraph 0111). In addition, it would have been obvious to the ordinary artisan that the known techniques of using the functionally equivalent hydrazones of Schwartz could have been combined with the method of Winther with predictable results because the known techniques of Winther and Schwartz predictably result in reliable immobilization of the amine-bearing molecule.
Neither Winther nor Schwartz teach reducing the potential for crosstalk by adding an unconjugated oligonucleotide.
However, Coull et al teach a method wherein addition of unconjugated (i.e., unlabeled) oligonucleotides having a sequence complementary to a probe (i.e., a mismatch; paragraph 0025; see also Figure 3). The unconjugated (i.e., unlabeled) is therefore complementary to the claimed detectable component (i.e., the labeled molecule of Yang in view of Schwartz).  Thus, because the purpose of the unconjugated (i.e., unlabeled) probe is to prevent nonspecific binding of two nucleotide sequences, the unlabeled probe could alternatively have been complementary to the molecular probe.  Coull et al also teach the addition of the unlabeled probe has the added advantage of reducing crosstalk (i.e., by increasing the signal to noise ratio by reducing non-specific binding) without substantially altering the detection limit of an assay (paragraph 0025).  Thus, Coull et al teach the known techniques discussed above.
With respect to the claimed washing and stripping step, paragraph 0541 of Example 28 of the instant specification states that stripping includes subjecting the sample “to conditions of temperature and ionic conditions sufficient to dchybridize [sic] the molecular probes’ oligonucleotide pairs.”  Thus, the claimed “stripping” merely encompasses dehybridization, and the claim has been given the broadest reasonable interpretation consistent with the teachings of the specification regarding “stripping” (In re Hyatt, 211 F.3d1367, 1372, 54 USPQ2d 1664, 1667 (Fed. Cir. 2000) (see MPEP 2111]).
Winther teaches target detection that utilizes heat denaturation and pH changes (paragraph 0004), which changes the ionic conditions.  Winther also teaches washing and altering the temperature, pH , or salt condition after detection (i.e., with previously detected) targets (paragraph 0071); altering pH and/or salt conditions alters the ionic conditions.
It is further noted that Winter teaches washing to remove hybridized components; i.e., use of stringent conditions, which includes washing, increased temperature, and altering ionic conditions by going from 5X SSC to 6X SSC (e.g., paragraphs 0171).
Coull et al also teach denaturing using ionic strength and temperature, as well as washing to remove unhybridized molecules (e.g., targets; paragraph 0351; see also paragraph 0282).  Thus, all of the steps for washing and stripping are taught by the cited prior art.
It is reiterated that the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.  Thus, any order of performing the steps, including washing and stripping after signal detection, is an obvious variant of the steps of the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record, and that the Response above should not be construed as an invitation to file an after final declaration.
It would therefore have been obvious to a person of ordinary skill in the art to have combined the method of Winther and Schwartz so as to include the unconjugated oligonucleotide of Coull et al to arrive at the instantly claimed method with a reasonable expectation of success.  The ordinary artisan would have been motivated to make the modification because said modification would have resulted in a method having the added advantage of reducing crosstalk by increasing the signal to noise ratio via reduction of non-specific binding without substantially altering the detection limit of an assay of as explicitly taught by Coull et al (paragraph 0025).  In addition, it would have been obvious to the ordinary artisan that the known techniques of Coull et al could have been applied to the method of Winther in view of Schwartz with predictable results because the known techniques of Coull et al predictably result in an improved signal to noise ratio.
Regarding claims 2-3, the method of claim 1 is discussed above.  Winther teaches the target is detected by binding the probe, and that labeling may be later than probe binding (paragraph 0004), thus, it would have been obvious to either bind the target to the binding moiety, followed by hybridizing the first and second oligonucleotides by subsequently adding the detection unit, or to bind probe and detectable component prior to target binding.
Alternatively, it is reiterated the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results, and that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.  Thus, any order of combining the components represents an obvious variant and/or routine optimization of the steps of the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record, and that the Response above should not be construed as an invitation to file an after final declaration.
Regarding claim 8, the method of claim 1 is discussed above.  Winther further teaches preparing each nucleic acid (e.g., PNA) probes that are purified by HPLC to give pure fractions (paragraph 0238).
With respect to the claimed purity, it is reiterated that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Therefore, the claimed purity merely represents routine optimization of the purification taught by the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record, and that the Response above should not be construed as an invitation to file an after final declaration.
Regarding claims 9-10, the method of claim 1 is discussed above.  Winther teaches purification of an oligonucleotide (e.g., PNA) using HPLC, which retards the oligonucleotide and allows separation from other reagents (Example 9).  Schwartz also teaches wash steps (paragraph 0204), as well as HPLC purification (paragraph 0219).  Coull et al teach conjugation of a binding moiety (i.e., enzyme) with an oligonucleotide followed by washing (paragraph 0176), as well as HPLC purification (paragraph 0159).  Thus, all of the claimed steps are taught by the prior art.
It is reiterated that the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.  Thus, any order of combining the components is an obvious variant of the steps of the prior art.
With respect to the claimed purity, Applicant is reminded that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Therefore, the claimed purity merely represents routine optimization of the purification taught by the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record, and that the Response above should not be construed as an invitation to file an after final declaration.
Regarding claim 11, the method of claim 1 is discussed above.  Schwartz et al teach conjugating with molecules having carbonyl groups (paragraphs 0145-0148); Winther teaches aldehydes (paragraph 0186), which have a carbonyl group.  Schwartz also teaches wash steps (paragraph 0204), as well as HPLC purification (paragraph 0219).  Winther teaches purification of an oligonucleotide (e.g., PNA) using HPLC, which retards the oligonucleotide and allows separation from other reagents (Example 9).  Coull et al teach washing (paragraph 0176), as well as HPLC purification (paragraph 0159).  Thus, all of the claimed steps are taught by the prior art.
It is reiterated that the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.  Thus, any order of combining the components is an obvious variant of the steps of the prior art.
With respect to the claimed purity, Schwartz teaches greater than 80% formation of the conjugate (paragraph 0216).
It is noted that the courts have stated where the claimed ranges “overlap or lie inside the ranges disclosed by the prior art” and even when the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have similar properties, a prima facie case of obviousness exists (see In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990); Titanium Metals Corp. of America v. Banner, 778 F2d 775. 227 USPQ 773 (Fed. Cir. 1985) (see MPEP 2144.05.01).
Applicant is also reminded that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Therefore, the claimed purity merely represents routine optimization and/or an obvious variant of the purity taught by the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record, and that the Response above should not be construed as an invitation to file an after final declaration.
Regarding claim 13, the method of claim 1 is discussed above.  Winther teaches the scaffold is a polymer scaffold (Figures 3 and 6, and paragraphs 0013 and 0016), which comprises a polysaccharide, in the form of the polysaccharide dextran or a dendrimer (paragraphs 0174-0175).
Regarding claim 14, the method of claim 1 is discussed above.  Winther teaches the antibody is a monoclonal antibody (paragraph 0123).
Regarding claims 15-16 and 21-22, the method of claim 1 is discussed above.  Winther teaches the target is an antigen or a protein (paragraph 0087), and that the antibody is a monoclonal antibody (paragraph 0123).
Regarding claim 17, the method of claim 11 is discussed above.  Winther teaches the labels are fluorescent dyes (paragraph 0017).
Regarding claim 18, the method of claim 1 is discussed above.  Winther teaches the method is automated (Abstract).
Regarding claim 23, the method of claim 1 is discussed above.  Winther teaches lengths of 10 bases (Table 8), which is in the claimed range.
In addition, it is reiterated that the courts have stated that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation, and that the courts have stated where the claimed ranges overlap or lie inside the ranges disclosed by the prior art and even when the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have similar properties, a prima facie case of obviousness exists.
Therefore, the claimed ranges merely represent an obvious variant and/or routine optimization of the lengths taught by the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record, and that the Response above should not be construed as an invitation to file an after final declaration.
Regarding claims 24-25, the method of claim 1 is discussed above.  Winther teaches the target is detected by binding the probe, and that labeling may be later than probe binding (paragraph 0004); thus, it would have been obvious to bind probe and detectable component, which bind to each other via hybridization, prior to target binding.
Alternatively, it is reiterated the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results, and that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.  Thus, any order of combining the components represents an obvious variant and/or routine optimization of the steps of the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record, and that the Response above should not be construed as an invitation to file an after final declaration.
Regarding claims 25-28, the method of claim 1 is discussed above.
It is reiterated the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results, and that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.  Thus, any order of combining the components represents an obvious variant and/or routine optimization of the steps of the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record, and that the Response above should not be construed as an invitation to file an after final declaration.
Regarding claims 29-30, the method of claim 1 is discussed above.  Winther teaches the signal generator comprises multiple detectable labels, which are attached via a polymer scaffold (Figures 3 and 6, and paragraphs 0013 and 0016), and that polymer comprises a polysaccharide, in the form of the polysaccharide dextran (paragraphs 0174-0175).
10.	Claims 1-3, 8-11, 13-18, and 21-30 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) based on the citations provided above.
11.	Claim 7 is rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Salazar et al (U.S. Patent Application Publication No. US 2009/0051912 A1, published 26 February 2009).
Regarding claim 7, the method of claim 1 is discussed above in Section 9.
While Winther also teaches multiplex assays (i.e., simultaneous detection of two conjugates; paragraph 0115), as well as binding detection using microscopy (paragraph 0091), neither Winther, Schwartz, nor Coull et al explicitly teach the combination of flow cytometry and microscopy.
However, Salazar et al teach 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 (paragraph 0051).  Thus, Salazar et al teach the known techniques discussed above.
It would therefore have been obvious to a person of ordinary skill in the art to have combined the method of Winther, Schwartz, and Coull et al with the teachings of Salazar et al to arrive at the instantly claimed method with a reasonable expectation of success.  The ordinary artisan would have been motivated to make the combination because said combination would have resulted in a method having the added advantage of allowing analysis of the sizes and attributes of cells without having to replace major components of a flow cytometer as explicitly taught by Salazar et al (paragraph 0051). In addition, it would have been obvious to the ordinary artisan that the known techniques of Salazar et al could have been combined with the method of Winther, Schwartz, and Coull et al with predictable results because the known techniques of Salazar et al predictably result in a reliable combination of components for cell detection.
12.	Claim 7 is rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Salazar et al (U.S. Patent Application Publication No. US 2009/0051912 A1, published 26 February 2009) based on the citations provided above.
13.	Claims 29-31 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Rowley et al (U.S. Patent No. 4,975,532, issued 4 December 1990).
It is noted that while claims 29-30 are rejected as described above, the claims are also obvious using the interpretation outlined below.
Regarding claims 29-31, the method of claim 1 is discussed above in Section 9.
As noted above, Winther teaches the oligonucleotide within the recognition unit and the oligonucleotide within the detection units are each covalently attached (paragraph 0174), as well as the use of hydrazines (paragraph 0078) and aldehydes (paragraph 0186), which form hydrazones, and that the polymer comprising the labels is dextran (paragraphs 0174-0175).
Schwartz teaches crosslinking using a hydrazine moiety to link an amino group (Abstract) to form a hydrazine (paragraph 0028).
Rowley et al teach methods wherein dextran is linked to fluorescent labels using hydrazone linkages (column 7, lines 1-20).  Rowley et al also teach the method has the added advantage of allowing direct labeling with multiple labeling entities while overcoming the problem of fluorescence quenching (column 2, lines 35-45).  Thus, Rowley et al teach the known techniques discussed above.
It would therefore have been obvious to a person of ordinary skill in the art to have combined the method of Winther, Schwartz, and Coull et al with the teachings of Rowley et al to arrive at the instantly claimed method with a reasonable expectation of success.  The ordinary artisan would have been motivated to make the combination because said combination would have resulted in a method having the added advantage of allowing direct labeling with multiple labeling entities while overcoming the problem of fluorescence quenching as explicitly taught by Rowley et al (column 2, lines 35-45). In addition, it would have been obvious to the ordinary artisan that the known techniques of Rowley et al could have been combined with the method of Winther, Schwartz, and Coull et al with predictable results because the known techniques of Rowley et al predictably result in a reliable labeling of dextran.
14.	Claims 29-31 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Rowley et al (U.S. Patent No. 4,975,532, issued 4 December 1990) based on the citations provided above.
Double Patenting
15.	The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA  as explained in MPEP § 2159.  See MPEP §§ 706.02(l)(1) - 706.02(l)(3) for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/process/file/efs/guidance/eTD-info-I.jsp.
16.	Claims 1-3, 8-11, 13-18, 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 (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003).
Both sets of claims are drawn to the same limitations; e.g., reducing cross talk using a molecular probe comprising a binding moiety having a specific binding affinity range, covalent linkages including hydrazones, hybridization, detection using flow cytometry, etc.  Any additional limitations of the ‘936 claims are encompassed by the open claim language “comprising” found in the instant claims.
Any missing limitations are found in the cited references as noted in the obviousness rejections under 35 U.S.C. 103(a) as detailed above.
17.	Claims 1-3, 8-11, 13-18, 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 (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) based on the citations presented above.
18.	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 (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Salazar et al (U.S. Patent Application Publication No. US 2009/0051912 A1, published 26 February 2009) based on the citations provided above.
19.	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 (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Salazar et al (U.S. Patent Application Publication No. US 2009/0051912 A1, published 26 February 2009) based on the citations provided above based on the citations presented above.
20.	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 (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Rowley et al (U.S. Patent No. 4,975,532, issued 4 December 1990) based on the citations provided above.
21.	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 (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Rowley et al (U.S. Patent No. 4,975,532, issued 4 December 1990) based on the citations provided above.
Response to Arguments
22.	Applicant’s arguments filed 19 December 2023 (hereafter the “Remarks”) have been fully considered but they are not persuasive for the reasons discussed below.
A.	Pages 10-11 of the Remarks summarize the amendments and include the interview summary discussed above.
B.	Pages 11-13 of the Remarks summarize Applicant’s interpretation of the cited prior art, and discuss the amendments to claim 1.
C.	Applicant argue on pages 13-14 of the Remarks that Winther does not teach the claimed washing and stripping.
However, as noted above, paragraph 0541 of Example 28 of the instant specification states that stripping includes subjecting the sample “to conditions of temperature and ionic conditions sufficient to dchybridize [sic] the molecular probes’ oligonucleotide pairs.”  Thus, the claimed “stripping” merely encompasses dehybridization, and the claim has been given the broadest reasonable interpretation consistent with the teachings of the specification regarding “washing and stripping.”
Winther teaches target detection that utilizes heat denaturation and pH changes (paragraph 0004), which changes the ionic conditions.  Winther also teaches washing and altering the temperature, pH , or salt condition after detection (i.e., with previously detected) targets (paragraph 0071); altering pH and/or salt conditions alters the ionic conditions.
It is further noted that Winter teaches washing to remove hybridized components; i.e., use of stringent conditions, which includes washing, increased temperature, and altering ionic conditions by going from 5X SSC to 6X SSC (e.g., paragraphs 0171).
Coull et al also teach denaturing using ionic strength and temperature, as well as washing to remove unhybridized molecules (e.g., targets; paragraph 0351; see also paragraph 0282).  Thus, all of the steps for washing and stripping are taught by the cited prior art.
It is reiterated that the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.  Thus, any order of performing the steps, including washing and stripping after signal detection, is an obvious variant of the steps of the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record, and that the Response above should not be construed as an invitation to file an after final declaration.
D.	Applicant argues on page 14 of the Remarks that Winther teaches crosslinking.
However, it is noted that a reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill the art, including nonpreferred embodiments (see MPEP § 2123).  Thus, the teaching of paragraph 0002 of Winther (cited by Applicant in support of this argument), which states that targets “may” be distinguished by addition of a crosslinking agent encompasses alternate embodiments wherein crosslinking agents are not present (see also paragraphs 0006, 0011, 0016, which state that the crosslinker is present in “some embodiments,” “may be employed,” and “could be used,” respectively).
E.	Applicant’s argument on page 14 of the Remarks regarding crosslinking after hybridization is clearly hypothetical, and offers no evidence in support of the argument.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record, and that the Response above should not be construed as an invitation to file an after final declaration.
F.	Applicant argue on page 14 of the Remarks that Coull et al teaches away from the claimed invention, allegedly because Coull et al teach probes complementary to non-targets.
However, as noted in the rejections above, Coull et al teach addition of unconjugated (i.e., unlabeled) oligonucleotides having a sequence complementary to a probe (i.e., a mismatch; paragraph 0025; see also Figure 3). The unconjugated (i.e., unlabeled) is therefore complementary to the claimed detectable component (i.e., the labeled molecule of Yang in view of Schwartz).  Thus,  because the purpose of the unconjugated (i.e., unlabeled) probe is to prevent nonspecific binding of two nucleotide sequences, the unlabeled probe could alternatively have been complementary to the molecular probe.
G.	Applicant’s remaining arguments rely on alleged deficiencies previously addressed, which are unpersuasive for the reasons discussed above.  Therefore, the claims remained rejected based on the prior art citations presented in the rejections.
Conclusion
23.	No claim is allowed.
24.	THIS ACTION IS MADE FINAL.  Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
25.	A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action.  In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action.  In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
26.

APPLICANT REBUTTAL: REMARKS
Claims 1-3, 9, 26 and 28 have been amended. Claim 19 has been canceled. Claims 1-3, 7-11, 13-18, and 21-31 will be pending upon entry of this amendment.
Support for the amendment to claim 1 can be found in at least Example 28 (paragraphs [0620] - [0623] of the specification as published).
No new matter is being added with these amendments.
Appli
cant Initiated Interview Summary
Applicants thank the Examiner for the courteous video interview conducted on
December 7, 2023 with the undersigned attorney, patent scientist Claire Venard, Ph.D. and the assignee's representative, Benjamin Sodey, Senior Corporate Patent Counsel at MilliporeSigma.
Prior to the interview, Applicants' representative submitted informally a proposed amendment to claim 1 adding the further requirement "wherein the hybridized detectable component is removable from the bound target by a stripping and washing process."
Applicants' representatives argued that the primary reference, Winther (WO
2007/031874 A2), makes use of a cross-linking agent that can impede removing hybridized components and is distinguished by the proposed amendment to claim 1. The Examiner noted that the disclosure in Winther does not appear to require use of a cross-linking agent and that the proposed claim amendment does not require that the hybridized detectable component be removed from the bound target. The Examiner suggested that making the proposed amendment to claim 1 into an active step of the claimed process in which the hybridized detectable component is required to be removed from the bound target. The disclosure of Winther at paragraph [0168] cited in the Non-Final Office dated August 10, 2023 against the features of dependent claim 19 was also discussed. The Examiner advised that he would have to review this disclosure more closely upon receipt of Applicants' response.
Applicants' representatives also argued that the cited reference Coull (US
2003/0124521), although teaching a technique for reducing cross-talk, uses an unlabeled probe complementary to a non-target sequence, which is distinguished by the requirement of
step (iv) in claim 1. The Examiner advised he would recheck the cited disclosure Coull against the asserted distinction upon receipt of Applicants' response.
No agreement was reached as to the pending claims or the proposed amended claims and the Examiner noted that further examination of the application will be dependent on an updated search.
The Applicants' representatives appreciate the preparation and consideration by the Examiner and the helpful suggestion of a claim amendment to advance prosecution.
Rej
ections Under 35 U.
S.C &103
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 A1), and Coull (US 2003/0124521).
The Office argues that Winther teaches a method of detecting a target in a sample using flow cytometry, comprising providing to the sample a molecular probe, in the form of a recognition unit, having a first oligonucleotide sequence, in the form of a nucleic acid segment, conjugated through a linker to a binding moiety, in the form of an antibody. The Office argues that Winther further teaches a detectable component, in the form of a detection unit, which comprises a second oligonucleotide (i.e., nucleic acid analog), which hybridizes to the first oligonucleotide sequence via a complementary sequence, and which further comprises a signal generator, in the form of multiple detectable labels, which are attached to a polymer scaffold. The Office argues that the polymer comprises a polysaccharide, in the form of the polysaccharide dextran or a dendrimer. The Office further contends that Winther also teaches the labels are fluorescent dyes, so none of the components are immobilized on solid supports. The Office argues that Winther further teaches the target is detected by binding the probe, and that labeling is later than probe binding, so it would have been obvious to bind the target to the binding moiety, followed by hybridizing the first and second oligonucleotides by subsequently adding the detection unit.
The Office admits that Winther does not teach the use of the hydrazone group. The Office adds Schwartz for teaching a method wherein a biomolecule is attached (i.e., cross-
linked) using a hydrazine moiety to link an amino group to form a hydrazine. The Office argues that it would have been obvious to combine the method of Winther with the teachings of Schwartz, which utilizes a functionally equivalent amine derived hydrazine, to arrive at the instantly claimed method with a reasonable expectation of success.
The Office further admits that neither Winther nor Schwartz teach reducing the
potential for cross-talk by adding an unconjugated oligonucleotide. The Office argues that Coull teaches a method wherein addition of unconjugated (i.e., unlabeled) oligonucleotides having a sequence complementary to a probe (i.e., a mismatch). The Office argues that the unconjugated oligonucleotide is therefore complementary to the claimed detectable component, and because the purpose of the unconjugated probe is to prevent nonspecific binding of two nucleotide sequences, the unlabeled probe could alternatively have been complementary to the molecular probe. The Office adds that Coull also teaches the addition of the unlabeled probe has the added advantage of reducing cross-talk. Thus, the Office argues that it would have been obvious to one of ordinary skill in the art to have combined Winther and Schwartz to include the unconjugated oligonucleotide of Coull to arrive at the claimed method with a reasonable expectation of success.
Regarding claim 19, the Office argues that Winther teaches washing to remove hybridized components; i.e. use of stringent conditions (paragraph [0168]).
Claim 1 as amended recites a flow cytometry method for detecting a target of a sample, comprising:
i)    mixing the sample with a molecular probe and a detectable 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; and b. 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; and b. 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.
Specifically, claim 1 has been amended to include a new limitation derived from now cancelled claim 19. Claim 1 now requires a further step vi) removing the hybridized detectable component from the bound target, wherein the removal is by a stripping and washing process.
While the Office argues that Winther teaches washing to remove hybridized
components; i.e. use of stringent conditions in paragraph [0168], Applicants respectfully disagree. While several paragraphs of Winther do refer to stringency (see paragraphs [0165] and [0168] - [0172]), use of stringent conditions is not a stripping and washing process after hybridization as required by the instant claims. Stringency generally refers to the hybridization step itself and how strict the pairing between the nucleotide pairs of nucleic acids has to be. For example, Winther refers to using nucleic acids that comprise sequences allowing hybridization to take place at a desired stringency (paragraph [0165]) and using conditions are such that sequences at least 70%, at least 80%, or at least 85-90% complementary nucleic acids remain bound to each other (paragraph [0168]). Similarly,
Schwartz and Coull are also silent on removing hybridized components as required by amended claim 1.
Applicants further argue that one would not be motivated to modify Winther to arrive at this limitation because Winther teaches a cross-linking step that can impede removing hybridized components. Winther discloses the importance of adding at least one cross-linking agent during the method to allow for drastic changes in buffer conditions or temperature to refine a detection procedure with minimal loss of signal (paragraph [0002]). For example, if the cross-linking agent is added after hybridization occurs, this would fix the hybridized detectable component in place and prevent stripping.
Moreover, Applicants argue that Coull teaches away from the reduction of cross-talk as claimed in the instant invention. Coull's reduction of cross-talk is directed to hybridization assays, but specifically to preventing probes binding non-target sequences (paragraphs [0022]). Coull teaches probes that are not completely complementary to the probe they are meant to block cross-talk for, unlike the present invention. For example, paragraph [0025] of Coull discloses the described surprising finding that:
the signal caused by the nonspecific binding of detectable probes to one or more non- target nucleic acid sequences can be dramatically suppressed by the addition of one or
more unlabeled probes wherein the sequence of the one or more unlabeled probes is
complementary to one or more non-target sequences to which the detectable probe binds
in a nonspecific manner.
Thus, Coull would teach using an unlabeled probe complementary to a non-target sequence, not the first or second oligonucleotide like step iv) of instant claim 1.
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 under 35
U.S.C. 103(a) as being unpatentable over Winther, Schwartz, and Coull as applied to claim
1 above and in further view of Salazar (US 2009/0051912).
The Office maintains the arguments concerning claim 1 above, but admits that
neither Winther, Schwartz, nor Coull explicitly teach the combination of flow cytometry
and microscopy. The Office adds that Salazer 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 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 Salazar to arrive at the instantly claimed method with a reasonable expectation of success.
Applicants maintain the arguments above concerning claim 1 and Winther,
Schwartz, and Coull and argues that Salazar fails to overcome these defects, as it is silent on removing hybridized components and reducing the potential for oligonucleotide cross- talk as required by amended claim 1.
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, Schwartz, and Coull as applied to claim
1 above and in further view of Rowley (US 4975532).
The Office maintains the arguments concerning claim 1 above. The Office adds that Rowley teaches 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 and Winther,
Schwartz, and Coull and argues that Rowley fails to overcome these defects, as it is silent on removing hybridized components and reducing the potential for oligonucleotide cross- talk as required by amended claim 1.
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 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

NEXT 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.

CITED REFERENCE SUMMARIES: Ref WO2007031874A2: Claims:
WHAT IS CLAIMED IS:
1. An automated method of detecting at least one target in a sample, comprising: a) obtaining a sample comprising at least one target; b) contacting the sample with at least one probe specific for the at least one target; c) contacting the sample with at least one detectable label; and d) detecting the presence of the at least one target with the at least one detectable label; wherein, following one or more of parts (a)-(c), the sample is incubated with at least one cross-linking agent; and wherein progress from one or more of parts (a)-(d) is automatically controlled.
2. The method of claim 1 , wherein the sample is incubated with the at least one cross-linking agent between parts (b) and (c).
3. The method of claim 2, wherein, following addition of the cross- linking agent between parts (b) and (c), buffer conditions of the sample are changed to conditions that without addition of the cross-linking agent would significantly reduce signal generated from the first target.
4. The method of claim 1 , wherein the sample is incubated with the at least one cross-linking agent following parts (c) and (d).
5. The method of claim 4, wherein, following addition of the cross- linking agent between parts (c) and (d), buffer conditions of the sample are changed to conditions that without addition of the cross-linking agent would
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significantly reduce signal from either or both of the at least one first target and at least one second target.
6. The method of claim 1 , wherein the at least one first target and the at least one second target comprise nucleic acids.
7. The method of claim 1 , wherein the at least one first target and the at least one second target comprise proteins.
8. The method of claim 1 , wherein the at least one first target is a protein and the at least one second target is a nucleic acid.
9. The method of claim 1 , wherein the at least one first target is a nucleic acid and the at least one second target is a protein.
10. The method of claim 1 , wherein the cross-linking agent covalently attaches the sample to at least one carrier.
11. The method of claim 1 , wherein the at least one first probe and/or at least one second probe is an antibody.
12. The method of claim 1 , wherein the at least one first probe and/or at least one second probe is a nucleic acid.
13. The method of claim 12, wherein the nucleic acid is a nucleic acid analog.
14. The method of claim 1 , comprising at least one first detectable label to detect the at least one first target and at least one second detectable label to detect the at least one second target, wherein the at least one first detectable label and the at least one second detectable label are distinguishable from each other.
15. An automated method of detecting a target in a sample, comprising:
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specific staining was reduced from 1+ to 0. Reintroducing 2 D's in D13155 instead of 2 A's (D13148) increased the non-specific staining from 1.5+ to 2.0+ (at 0.2 μM PNA). Both D12120 and D13155 had 2 D's in the sequence, but the non-specific staining in D13155 did not reach the same level as in D12120, probably due to the 2 G3 1S in D13155. The same effect of non-natural bases was seen when comparing D12120 with D14133: the non-specific staining in D14133 did not reach the same level as in D12120, this time probably due to 2 inosines (l)'s in D14133. The replacement of 2 G's in D13122 with 2 Gs's (D13159) reduced the non-specific staining from 3+ to 0 (at a PNA concentration of 0.2 μM). Substitution of 1 G in D14110 with 1 Gs (D14089), reduced the non-specific staining from 1+ to 0. Equally, 1 G in D14086 was replaced with 1 G8 (D14089), resulting in a reduction of the non-specific staining from 3+ to 0 (at a PNA concentration of 0.2 μM).
[0149] The non-specific background staining generated by the PNA2 conjugates could be subtly fine tuned, and ultimately completely eliminated, by base substitution using both natural as well as non-natural bases. The level of background correlated directly to the DNA/RNA affinity of the PNAs of the conjugates. This was surprising, as the conjugates have a molecular weight around 500 kDa, and the changes in the PNAs necessary to bring about strongly reduced background in some cases were as little as one D to A substitution (thus removing one potentially hydrogen bonding amino group) or one G to G8 substitution (introducing a single carbonyl to thiocarbonyl change).
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 Table 21 : Non-specific staining intensities of due to PNA2 conjugates.
Example 35. Use of fluorescein as a molecular label
[0150] Tonsil tissues were taken through the steps 1-4, 6-8, 10, and 13 (PNA2 was conjugated with fluorescein) and 16 (the slides were mounted with Vectashield containing DAPI). The concentration used for the conjugates was 0.08μM for PNA1 , D13171 and 0.02, 0.05, 0.1 , and 0.2μM for PNA2, D14008 (TCD DGSGSTAC A). The slides were evaluated using a fluorescence microscope at 4Ox and 100x magnifications.
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 [0151] The specific staining was satisfactory. This experiment demonstrated the possibility of making PNA-fluorescein conjugates and illustrated the application of the present invention with a fluorescein as detectable label.
Example 36. Test of a PNA pair with 10 antibodies
[0152] A multi-block containing tissue from mammalian carcinoma, kidney, colon and two tonsils was taken through the steps 3-4, 6-8, 10, 13-17. In step 8, ten different primary antibodies and a negative control mouse IgGI were used. (See table 22.) The concentration of the conjugates used was 0.08 μM for PNA1 , D12102 and 0.05 μM for PNA2, D12094. Visualization of primary reagents by the K5007 detection kit was performed in parallel according to the manufacturer's instructions. Two primary antibodies targeting membranous targets were used: Monoclonal Mouse Anti-Human CD20cy (DakoCytomation code No. M0755) and Monoclonal Mouse Anti-Human Epithelial Membrane Antigen (DakoCytomation code No. M0613). Five primary antibodies targeting cytoplasmic targets were used: Monoclonal Mouse Anti-Human Cytokeratin (DakoCytomation code No. M3515), Monoclonal Mouse Anti-Human Desmin (DakoCytomation code No. M0760), Monoclonal Mouse Anti-Human CD68 (DakoCytomation code No. M0876), Monoclonal Mouse Anti-Human BCL2 (DakoCytomation code No. 0887) and Monoclonal Mouse Anti-Human CD45 LCA (DakoCytomation code No. M0701). Three primary antibodies targeting nuclear targets were used: Monoclonal Mouse Anti-Human Estrogen Receptor (DakoCytomation code No. M7047), Monoclonal Mouse Anti-Human Ki-67 Antigen (DakoCytomation code No. M7240) and Monoclonal Mouse Anti-Human p27 (DakoCytomation code No. M7203). The primary antibodies, all products from DakoCytomation, were diluted in S2022 as indicated in Table 22.
[0153] This example shows the extended use of the PNA system employing several primary antibodies. Furthermore, in the majority of cases using the 10 primary antibodies, visualization by the PNA based detection system demonstrated an improved staining as compared to the reference K5007.
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 Table 22: Comparison of the PNA based detection system with En Vision TM
Example 37. Standard Synthesis of AP-DexVS70-PNA Conjugate
[0173] Alkaline Phosphatase ("AP") (from Calf Intestine, EIA grade) was dialyzed overnight against 2mM HEPES, pH 7.2; 0.1 M NaCI; 0.02mM ZnCI2. Dextran (molecular weight 7OkDa) was activated with divinylsulfone to a degree of 92 reactive groups per dextran polymer (DexVS70).
[0174] The three components below were mixed together and placed in a water bath at 400C for 30 minutes.
192.0 μL DexVS70 13.7 nmol 41.0 μL PNA 41 nmol PNA dissolved in H2O 6.0 μL 1 M NaHCO3
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 [0175] 108.0μL of the DexVS70-PNA conjugate was taken out and added to a mixture of:
160.0μl_ AP 43.4 nmol
7.7μL 1M NaHCO3
30.6μl_ 2OmM Hepes, pH 7.2; 1 M NaCI; 5OmM MgCl2; 1 mM ZnCI2
[0176] The mixture was placed in a water bath at 400C for 3 hours. Quenching was performed by adding 30.6 μl_ of 0.1 M ethanolamine and letting the mixture stand for 30 minutes in water bath at 400C. The product was purified on FPLC with: Column Superdex-200, buffer: 2mM HEPES, pH 7.2; 0.1 M NaCI; 5mM MgCI2; 0.1 mM ZnCI2. Two fractions were collected, one with the product and one with the residue. [0177] In comparison to the experiment described above, another conjugate was made with extended conjugation time. The three components below were mixed together and placed in a water bath at 400C for 30 minutes.
192.0μL DexVS70 13.7 nmol
41.OμL PNA 41 nmol PNA dissolved in H2O
6.0μL 1 M NaHCO3
[0178] 108.0μl_ of the DexVS70-PNA conjugate was taken out and added to a mixture of:
160.0μL AP 43.4 nmol
7.7μL 1 M NaHCO3 30.6μL 2OmM Hepes, pH 7.2; 1 M NaCI; 5OmM MgCI2; 1 mM ZnCI2
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 [0179] The mixture was placed in a water bath at 400C for 5 hours. Quenching was performed by adding 30.6μL 0.1 M Ethanolamine and letting the mixture stand for 30 minutes in water bath at 4O0C. Purification of the product on FPLC: Column Superdex-200, buffer: 2mM Hepes, pH 7.2; 0.1 M NaCI; 5mM MgCI2; 0.1 mM ZnCI2. Two fractions were collected: One with the product and one with the residue.
[0180] Relative absorbance PNA(FIu) (ε5OOnm= 73000M'1) and AP(ε278nm= 140000M"1. Corrected for absorbance from PNA at 278nm, this correction factor is due to the specific PNA and it is calculated: 278/500nm) was used to calculate the average conjugation ratio of PNA, AP and DexVS70.
AP-DexVS70-PNA, 3hrs: PNA/DexVS70: 1.8 AP/DexVS70: 1.8
AP-DexVS70-PNA, 5hrs: PNA/DexVS70: 2.0 AP/DexVS70: 2.4
[0181] Due to these results, it is recommended to follow a procedure in which the conjugation time (AP + DexVS70-PNA) is 5 hours. Example 36. Synthesis and IHC Testing of an Antibody-PNA Conjugate
Part A. Testing Different Ratios of Linker to Antibody
[01821 Materials: Antibody: CD 45 dialized overnight against 0.01 M Hepes 0.1 M NaCI pH=7.2. SMCC: Succinimidyl -4(N-maleimidomethyl) cyclohexan-1- carboxylate molw. 334.33. PNA: Acetyl- L30-GTP-TAA-TTP-PAG-L150-LyS(CyS) Test i
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 [0183] 10 nmol CD45 was dissolved in 161 μl_ 0.01 M Hepes 0.1 M NaCI pH=7.2. 250 nmol SMCC was dissolved in 8 μl_ NMP. The above components were mixed and placed in a water bath at 30° C for 60 minutes. The mixture was purified on a mini-prep column (Sephadex G-25) with 0.01 M Hepes 0.1 M NaCI pH=7.2 as eluent. Fractions of 0.3 ml_. By measuring absorbance at 278 nm three fractions containing the product (58 %) were identified. These three fractions were added to 100 nmol of a lyophilised PNA. Then 1 μL 5 % Di-Sodium-EDTA/water was added and the solution was mixed until dissolved and placed in a water bath at 30°C for 30 minutes. Quenching was performed by adding 2 mg of Cysteine. Water bath 30°C for 30 minutes.
[0184] The product was purified on FPLC: Column SUPERDEX®-75, Buffer 0.01 M Hepes 0.1 M NaCI pH 7.2. The fraction with the product was collected. Relative absorbance between PNA (ε260 nm) and antibody (s278 nm) was used to calculate the average conjugation ratio of PNA and antibody. PNA / CD45: 5.2. Yield 39 % based on antibody. Test 2
[0185] 10 nmol CD45 was dissolved in 161 μL 0.01 M Hepes 0.1 M NaCI pH=7.2. 150 nmol SMCC was dissolved in 5 μL NMP. The above components were mixed and placed in a water bath at 30° C for 60 minutes. The mixture was purified on a mini-prep column (SEPHADEX® G-25) with 0.01 M Hepes 0.1 M NaCI pH=7.2 as eluent. Fractions of 0.3 mL. By measuring absorbance at 278 nm three fractions containing the product (74 %) were identified.
[0186] These three fractions were added to 100 nmol of a lyophilized PNA. Then 1 μL 5 % Di-Sodium-EDTA/water was added and the solution was mixed until dissolved and placed in a water bath at 3O0C for 30 minutes. Quenching was performed by adding 2 mg of cysteine. Water bath 30°C for 30 minutes.
[0187] The product was purified on FPLC: Column Superdex-75, Buffer 0.01 M Hepes 0.1 M NaCI pH 7.2. The fraction with the product was collected. Relative absorbance between PNA (ε2eo nm) and antibody (ε278 nm) was used to calculate the
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average conjugation ratio of PNA and antibody. PNA / CD45: 3.4. Yield 55 % based on antibody. IHC Test of Conjugates
[0188] A later IHC test showed that PNA / CD45 in Test 2 gave a higher score than the one in Test 1. This brought us to the conclusion that the ratio between CD45 / SMCC / PNA should be 10 / 150 / 100.
Part B. Test of Different Conjugation Times - Antibody and Linker
[01891 Materials: Antibody: GAM (goat-anti-mouse) dialysed overnight against 0.1 M NaCI. SMCC: Succinimidyl -4(N-maleimidomethyl) cyclohexan-1- carboxylate molw. 334.33. Flu-Link: Flu-Lgo-Lys(Flu)-L3O-Lys(Cys) Test 1
20 nmol GAM was dissolved in 402 μL 0.01 M Hepes 0.1 M NaCI pH=7.2. 400 nmol SMCC dissolved in 13 μL NMP. The above components were mixed and placed in a water bath at 30° C for 1 hour. 207 μL of the mixture was purified on a mini-prep column (SEPHADEX® G-25) with 0.01 M Hepes 0.1 M NaCI pH=7.2 as eluent. Fractions of 0.3 mL were taken. By measuring absorbance at 278 nm, three fractions containing the product (79 %) were identified. These three fractions were added to 200 nmol of a lyophilized Flu-Link. Then 1 μL 5 % di-sodium-EDTA/water was added and the solution was mixed until dissolved and placed in a water bath at 30°C overnight. Quenching was performed by adding 2 mg of Cysteine. Water bath 30°C for 30 minutes.
[0190] The product was purified on FPLC: Column SUPERDEXΘ-75, Buffer 0.01 M Hepes 0.1 M NaCI pH 7.2. The fraction with the product was collected. Relative absorbance between Flu-Link (ε4g8 nm) and antibody (ε278nm) was used to calculate the average conjugation ratio of PNA and antibody. Flu-Link / GAM: 7.1. Yield 55 % based on antibody. Test 2
[0191] 20 nmol GAM was diluted with 402 μL 0.01 M Hepes 0.1 M NaCI pH=7.2. 400 nmol SMCC was dissolved in 13 μL NMP. The above components
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were mixed and placed in a water bath at 30° C for 2 hours. The rest of the synthesis and purification was done in exactly the same procedure as for 1 hour. There was a 64 % yield of GAM/SMCC before adding the Flu-Link. Flu-Link / GAM: 8.7. Yield 33 % based on antibody.
Part C. Test of Different Conjugation Times - Fluorophore and Linker
[01921 Materials: Antibody: GAM dialysed overnight against 0.1 M NaCI. SMCC: Succinimidyl -4(N-maleimidomethyl) cyclohexan-1-carboxylate molw. 334.33. Flu-Link: FIu-L90-LyS(FIu)-L3O-LyS(CyS) Test i
[0193] 20 nmol GAM was dissolved 378 μL 0.01 M Hepes 0.1 M NaCI pH=7.2. 400 nmol SMCC was dissolved in 13 μL NMP. The above components were mixed and placed in a water bath at 30° C for 1 hour. The mixture was divided in two and purified on two mini-prep columns (SEPHADEX® G-25) with 0.01 M Hepes 0.1 M NaCI pH=7.2 as eluent. Fractions of 0.3 mL were taken. By measuring absorbance at 278 nm, three fractions from each column containing the product (76 % in all) were identified. These six fractions were pooled, divided in two, and each added to 200 nmol of a lyophilized Flu-Link. Then 1 μL 5 % di-sodium-EDTA/water was added and the solution was mixed until dissolved and placed in a water bath at 30°C, one for 30 minutes, the other for 60 minutes. Quenching was performed by adding 2 mg of cysteine. Water bath 3O0C for 30 minutes.
[0194] The product was purified on FPLC: Column SUPERDEXΘ-75, Buffer 0.01 M Hepes 0.1 M NaCI pH 7.2. The fractions with the product from each purification were collected. Relative absorbance between Flu-Link (ε498 nm) and antibody (ε278nm) was used to calculate the average conjugation ratio of PNA and antibody. 30 minutes conjugation Flu-Link / GAM: 7.0 Yield 55 % based on antibody. 60 minutes conjugation Flu-Link / GAM: 6.9 Yield 52 % based on
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antibody. The above results show that 30 minutes conjugation between GAM /
SMCC and Flu-Link is sufficient.
Example 39. Additional Tests of 2 and 3 Layer Visualization Systems
[0195] Primary mouse antibody M7240 (Dako) targeting MIB-1 was diluted to final 1 :150 in S2022 buffer (Dako) and applied on a multi tissue section. Following 10 minutes incubation at RT the section was washed 5 minutes using 10x diluted S3006 buffer (Dako).
[0196] Goat-anti-mouse secondary antibody conjugated with dextran and a first PNA sequence (GaM-dex-PNA1 (218-117)) was diluted to final concentration of 0.08 μM (based on dextran) in BP-HEPES-buffer (1.5% BSA, 3% PEG, 0.15M NaCI, 1 OmM HEPES, pH 7.2) and was applied to the section. Following 10 minutes incubation at room temperature (RT), the section was washed 5 minutes using 10x diluted S3006 (Dako). The sections were rinsed in deionized water. Following 10 minutes incubation in 0.5% glutaraldehyde at RT, the sections were rinsed in deionized water and washed 5 minutes using 10x diluted S3006 (Dako).
[0197] An adaptor unit comprising dextran coupled to two different PNA sequences, one complementary to PNA1 above (PNA2) and another not complementary to PNA1 (PNA3), called PNA2-dex-PNA3 (218-057) was diluted to a final concentration of 0.05 μM (dextran) in BP-HEPES-buffer (1.5% BSA, 3% PEG, 0.15M NaCI, 1OmM HEPES, pH 7.2) and was applied to the section. Following 10 minutes incubation at RT, the section was washed 5 minutes using 10x diluted S3006 (Dako). Next, a conjugate of a PNA4, complementary to PNA3 above, dextran, and the detectable label alkaline phosphatase (PNA4-dex-AP (209-177)) was diluted to final concentration of 0.05 μM (dextran) in BAP-HEPES-buffer (1.5% BSA, 3% PEG, 0.15M NaCI, 0.05% 4-aminoantipyrin, 1OmM HEPES, pH 7.2), and was applied. Following 10 minutes incubation at RT, the sections were washed 5 minutes using 10x diluted S3006 (Dako).
[0198] "Permanent Red working solution" (an aqueous Tris buffer with naphthol-phosphate and a diazonium dye; K0640 Dako) was prepared and then
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applied. Following 10 minutes incubation, the section was washed 5 minutes using 10x diluted S3006 (Dako). Finally the sections were counter stained 5 minutes using haematoxylin S3301 (Dako), rinsed in deionzed water, washed 3 minutes in wash buffer, and mounted in Faramount S3025 (Dako). Result: MIB-1 =1+/0.5+.
Example 40:
[0199] Primary rabbit antibody A0452 (Dako) targeting CD3 was diluted to final 1 :100 in S2022 (Dako) and applied on a multi tissue section. Following 10 minutes incubation at RT the sections were washed 5 minutes using 10x diluted S3006 (Dako).
[0200] Then, a goat-anti-rabbit secondary antibody coupled to dextran and a first PNA sequence, PNA2a (GaR-dex-Alexander (209-127)) was diluted to final concentration of 0.08 μM (dextran) in BP-HEPES-buffer (1.5% BSA, 3% PEG, 0.15M NaCI, 10 mM HEPES, pH 7.2) and was applied. Following 10 minutes incubation at RT the sections were washed 5 minutes using 10x diluted S3006 (Dako). The sections were rinsed in deionized water. Following 10 minutes incubation in 0.5% glutaraldehyde at RT the sections were rinsed in deionized water and washed 5 minutes using 10x diluted S3006 (Dako).
[0201] Next, a complementary PNA coupled to dextran and detectable label alkaline phosphatase (AP) (PNA2b-dex-AP (209-177) was diluted to final concentration of 0.05 μM (dextran) in BAP-HEPES-buffer (1.5% BSA, 3% PEG, 0.15M NaCI, 0.05% 4-aminoantipyrin, 1OmM HEPES, pH 7.2) and was applied. Following 10 minutes incubation at RT the section was washed 5 minutes using 10x diluted S3006 (Dako).
[0202] Permanent Red working solution (K0640 Dako) was prepared and was applied. Following 10 minutes incubation the sections were washed 5 minutes using 10x diluted S3006 (Dako). Finally the sections were counter stained 5 minutes using haematoxylin S3301 (Dako), rinsed in deionzed water, washed 3 minutes in wash buffer, and mounted in Faramount S3025 (Dako). Result: CD3 specific staining
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=1.5+ compared to non-specific background staining of 1 +. The order of detection affects the staining result.
Example 41. Detecting Two Targets in a Sample
[0203] General Procedural Note: Before conducting the dete
[Truncated to input limit]