DETAILED ACTION
The amendment filed on 10/14/2025 has been entered. No new matter has been added.
Claim 26 is amended in the claim set filed on 10/14/2025.
Claims 1-14 and 52-55 are canceled.
Claims 86-102 are added.
Claims 26 and 86-102 in the claim set filed on 10/14/2025 are pending and currently under examination.
Response to the Arguments
Objections to the Drawings in the previously mailed non-final are withdrawn in light of applicants Drawings amendments. Note: Further amendment to figures may be required if the claims become allowable.
The 35 U.S.C. 112 rejections documented in the previously mailed non-final have been withdrawn in light of applicants claim cancellations.
Applicant’s arguments regarding previous rejection(s) of claim(s) 1-14 under 35 U.S.C. 103 have been withdrawn in light of applicants claim cancellations.
Applicant’s arguments regarding previous rejection(s) of claim(s) 26 under 35 U.S.C. 103 have been fully considered and are persuasive. Applicant’s argument on Pg. 8, states that “a prima facie case of obviousness has not been established” and “claim 26 is directed to a method in which specific steps are conducted in a specific order. In particular, the sample is first incubated with a primary antibody, then treated with a crosslinking agent, then treated with a protease, then a target nucleic acid is detected by in situ hybridization, and then the target protein is detected by incubating the sample with a secondary antibody. This specific order is not disclosed in Winther, particularly with the inclusion of protease treatment and the incubation with the second antibody or other labeling method to detect the target protein after all other steps are completed.” The 35 U.S.C. 103 rejections documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 7-10. However, upon further consideration and search, new grounds of rejection for claims 26 and 86-102 under 35 U.S.C. 103 are documented below, in this office action on Pg. 3-15.
The rejections for claims 26 and 86-102, documented below in this Final Office Action, are necessitated by claim amendments filed on 10/14/2025.
Priority
This application is a 371 of PCT/US2021/031034 05/06/2021 which claims priority to and the benefit of U.S. Provisional Patent Application No. 63/021,632 filed on 05/07/2020. Accordingly, the priority date of instant claims is determined to be 05/07/2020, the filing date of U.S. Provisional Application No. 63/021,632.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 26 and 86-99 are rejected under 35 U.S.C. 103 as being unpatentable over Lars Winther (“Winther”; US Patent App. Pub. US 2010/0105145 A1, April 29, 2010).
Winther discloses optionally automated methods that may be used to qualitatively and/or quantitatively detect at least one, or for example, two or more different targets in a sample, and kits associated with such methods. The two or more different targets may be detected and distinguished by adding at least one cross-linking agent to the sample in between different steps of a detection procedure. The addition of a cross-linking agent may allow for drastic changes in buffer conditions (i.e. solvent, pH, salt concentration, etc.) or temperature in order to refine a detection procedure with minimal loss of signal. The instant invention is compatible with a variety of detection systems, including immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), flow cytometry, enzyme immuno-assays (EIA), enzyme linked immuno- assays (ELISA), blotting methods (e.g. Western, Southern, and Northern), labeling inside electrophoresis systems or on surfaces or arrays, and precipitation, among other general detection assay formats. The invention is also compatible with many different types of samples, targets, probes, and detectable labels.(Abstract)
Regarding claim 26, Winther teaches a method comprising "the sample is incubated with an optionally labeled primary antibody", "A cross-linking agent is added", "Following cross-linking… nucleic acid probe is added" and "the protein and gene targets are visualized via a color or fluorescent signal" (Para. 191). Winther teaches a method wherein “Some targets may require protease ... treatment” (Para. 4). Winter teaches a method wherein “the invention may be used in connection with … nucleic acid hybridization assays such as: … in situ hybridization (ISH)” (Para. 90). Winther teaches a method comprising “a primary antibody probe may be contacted with a secondary antibody conjugated to a detectable label” (Para. 113). Furthermore, Winter reiterates the method comprising steps (i-v), “Some embodiments of the instant invention include a method of detecting at least two targets in a sample, comprising: a) obtaining a sample comprising at least one first target and at least one second target; b) contacting the sample with at least one first probe specific for the at least one first target; c) contacting the sample with at least one second probe specific for the at least one second target; d) contacting the sample with at least one detectable label; and e) detecting the presence of the at least one first target and at least one second target with the at least one detectable label; wherein, following one or more of parts (a)-(d), the sample is incubated with at least one cross-linking agent.” (Para. 55-60) and wherein “at some stage after adding the cross-link, the buffer conditions of the sample may be altered to those which would have been incompatible with the detection process prior to cross-linking… addition of agents … such as protease” (Para. 70). “at some stage after adding the cross-link, the buffer conditions of the sample may be altered to … addition of agents… such as protease” is interpreted as adding protease after crosslinking. “the invention may be used in connection with … nucleic acid hybridization assays such as: … in situ hybridization (ISH)” reads on detecting the target nucleic acid by nucleic acid by in situ hybridization after (iii). “a primary antibody probe may be contacted with a secondary antibody conjugated to a detectable label” reads on detecting the target protein by incubating the biological sample with a secondary antibody or other labeling methods after (iv). Thus, Winther teaches a method for simultaneously detecting a target nucleic acid and a target protein in a biological sample, the method comprising:(i) incubating the biological sample with a primary antibody; (ii) treating the biological sample with a crosslinking agent after (i); (iii) treating the biological sample with a protease after (ii); (iv) detecting the target nucleic acid by in situ hybridization after (iii); and (v) detecting the target protein by incubating the biological sample with a secondary antibody or other labeling methods after (iv).
Therefore, the invention as recited in claim 26 is prima facie obvious over the prior art Winther et al. One of ordinary skill in the art would have had a reasonable expectation of success given the lack of novelty. It would have been obvious to provide a method for simultaneously detecting a target nucleic acid and a biological sample according to the limitations of the instant application claims 26 based on Winther et al. (US Patent App. Pub. US 2010/0105145 A1).
The teachings of Winther are documented above in the rejection of claim 26 under 35 U.S.C. 103. Claims 86-88, 90-95 and 98-99 depend on claim 26. Claim 89 depends on claim 88, which depends on claim 26. Claim 97 depends on claim 96, which depends on claim 95, which depends on claim 26.
Regarding claims 86-87, Winther teaches a method wherein “Methods of visualizing two or more different targets within the same sample… DNA or RNA sequence” (Para. 3) and “a nucleic acid segment refers to a nucleobase sequence comprising … formed solely from RNA or DNA nucleosides” (Para. 45). Thus, Winther teaches a method wherein the target nucleic acid is DNA or RNA.
Regarding claim 88, Winther teaches a method wherein “a nucleic acid or nucleic acid probe is added…Then, the sample is incubated with one or more detection conjugates” (Para.191). Winther teaches a method comprising detection of a nucleic acid as depicted in Figure 6, providing one or more target probe(s) capable of hybridizing to the target nucleic acid;(ii) providing a signal-generating complex capable of hybridizing to the one or more target probe(s), wherein the signal-generating complex comprises a nucleic acid component capable of hybridizing to the one or more target probe(s) and a label probe;(iii) hybridizing the target nucleic acid to the one or more target probe(s); and (iv) capturing the signal-generating complex to the one or more target probe(s) and thereby capturing the signal-generating complex to the target nucleic acid.(Fig. 6,far right, see below). Thus, Winther teaches a method wherein the step of detecting the target nucleic acid by in situ hybridization comprises:(i) providing one or more target probe(s) capable of hybridizing to the target nucleic acid;(ii) providing a signal-generating complex capable of hybridizing to the one or more target probe(s), wherein the signal-generating complex comprises a nucleic acid component capable of hybridizing to the one or more target probe(s) and a label probe;(iii) hybridizing the target nucleic acid to the one or more target probe(s); and(iv) capturing the signal-generating complex to the one or more target probe(s) and thereby capturing the signal-generating complex to the target nucleic acid.
PNG
media_image1.png
737
556
media_image1.png
Greyscale
Regarding claim 89, Winther teaches a method as depicted in Figure 6 above, wherein one or more target probe(s) comprises a target (T) section and a label (L) section, wherein the T section is a nucleic acid sequence complementary to a section on the target nucleic acid and the L section is a nucleic acid sequence complementary to a section on the nucleic acid component of the signal- generating complex, and wherein the T sections of the one or more target probe(s) are complementary to non-overlapping regions of the target nucleic acid, and the L sections of the one or more target probe(s) are complementary to non-overlapping regions of the nucleic acid component of the generating complex. (Figure 6, far right, see above) Thus, Winther teaches a method wherein each of the one or more target probe(s) comprises a target (T) section and a label (L) section, wherein the T section is a nucleic acid sequence complementary to a section on the target nucleic acid and the L section is a nucleic acid sequence complementary to a section on the nucleic acid component of the signal- generating complex, and wherein the T sections of the one or more target probe(s) are complementary to non-overlapping regions of the target nucleic acid, and the L sections of the one or more target probe(s) are complementary to non-overlapping regions of the nucleic acid component of the generating complex.
PNG
media_image2.png
794
882
media_image2.png
Greyscale
Regarding claim 90, Winther teaches a method wherein “target antigen bound to a primary antibody is recognized by a recognition unit comprising a secondary antibody probe. The recognition unit is specifically hybridized to a detection unit via the nucleic acid analog segments on each unit.” (Para.14; Fig. 4 see below). Thus, Winther teaches a method wherein the method further comprises providing an immunohistochemistry label capable of binding to the secondary antibody for detecting the target protein; or wherein the secondary antibody is pre-labeled.
Regarding claims 91-94, Winther teaches a method wherein “Samples may also be derived from living matter taken from any living organism…Samples may comprise a cell sample, such as a cell smear or colony … or a tissue specimen... They may also comprise a biological fluid, such as an animal-derived fluid, e.g. … whole blood” (Para. 86). Thus, Winther teaches a method wherein the biological sample is a tissue specimen or is derived from a tissue specimen, a blood sample, a cytological sample and/or cultured cells or a sample containing exosomes.
Regarding claims 95-97, Winther teaches a method wherein “the cross-linking agent may also be mixed with or may comprise tissue fixation reagents such as 4% formaldehyde” (Para. 83) and “neutral buffered formalin” (Para. 99). Formalin is interpreted as 37% formaldehyde. 10 % buffered formalin is interpreted as 4% formaldehyde. Thus, Winther teaches a method wherein the crosslinking agent is a fixative; wherein the fixative is neutral buffered formalin; wherein the neutral buffered formalin is 10% neutral buffered formalin.
Regarding claim 98, Winther teaches a method wherein “cross-linking agent may also be employed, such as one that is chemically reactive enough to form appropriate cross-links within minutes of application” (Para.82). “reactive enough to form appropriate cross-links within minutes of application” is interpreted as a treatment that lasts for at least 1-60 minutes. Thus, Winther teaches a method wherein the step of treating the biological sample with the crosslinking agent lasts for about 15 minutes, about 30 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.
Regarding claim 99, Winther teaches a method wherein “samples were fixed … The samples were incubated overnight in a ventilated laboratory hood at room temperature.” (Para. 279). Thus, Winther teaches a method wherein the step of treating the biological sample with the crosslinking agent is performed at about 4 room temperature, about 40 °C or about 60 °C.
Therefore, the invention as recited in claims 26 and 86-99 is prima facie obvious over the prior art Winther et al. One of ordinary skill in the art would have had a reasonable expectation of success given the lack of novelty. It would have been obvious to provide a method for simultaneously detecting a target nucleic acid and a biological sample according to the limitations of the instant application claims 26 and 86-99 based on Winther et al. (US Patent App. Pub. US 2010/0105145 A1).
Response to Arguments
Applicant's arguments filed 10/14/2025 (Pg. 7-10) with respect to claims 1-14 and 26 have been considered and are persuasive. To clarify some instances argued in the response filed 10/14/2025 see responses to each argument made by Applicant below:
Applicants’ argument: “the Office Action did not present any formal obviousness analysis, such as a discussion of how the present claims differ from what is taught in Winther, or why one skilled in the art would have been motivated to modify the teachings of Winther or reasonably expected success in doing so.” (Pg. 7)
Response: Applicant's arguments (Pg. 7) filed 10/14/2025 has been fully considered and persuasive. To clarify some instances argued in the response filed 07/08/2025 see responses to each argument made by Applicant below:
Applicants’ argument: “This specific order is not disclosed in Winther, particularly with the inclusion of protease treatment and the incubation with the second antibody or other labeling method to detect the target protein after all other steps are completed.” (Pg. 8)
Response: Applicant's argument filed 10/14/2025 has been fully considered but is not persuasive. Applicant's arguments filed 05/21/2025 do not apply to the new grounds of rejections.
Applicants’ argument: “Nowhere is a protease mentioned in this workflow, nor is a secondary antibody expressly disclosed. Rather, "one or more adaptor molecules that recognizes the primary antibody" are used before cross-linking." (Pg. 9)
Response: Applicant's arguments filed 10/14/2025 has been fully considered but are not persuasive. As recited in the Non-final office action and documented above in the new grounds of rejection, “Winther teaches a method wherein “at some stage after adding the cross-link, the buffer conditions of the sample may be altered to those which would have been incompatible with the detection process prior to cross-linking… addition of agents … such as protease” (Para. 70).This statement reads on step (iii) treating the biological sample with a protease. Furthermore, “at some stage after adding the crosslink” reads on the workflow recited previously. Nevertheless, it also is directed to para. 56-60 in the new grounds of rejection (see above). As recited in the Non-final office action and documented above in the new grounds of rejection, Winther teaches a method comprising “a primary antibody probe may be contacted with a secondary antibody conjugated to a detectable label” (Para. 113). “one or more adaptor molecules that recognizes the primary antibody" are used before cross-linking” is not meant to compensate for the secondary antibody. The language of the claim reads on an open-ended method and does not exclude additional, unrecited elements or method steps. See MPEP 2111.03. Thus, one skilled in the art would have reasonably expected such results based on the disclosure of Winther.
Claims 26 and 100 is rejected under 35 U.S.C. 103 as being unpatentable over Lars Winther (“Winther”; US Patent App. Pub. US 2010/0105145 A1, April 29, 2010 in view of Grabinski, et al. (“Grabinski”; (2015). A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures. PloS one, 10(3), e0120120.
The teachings of Winther are documented above in the rejection of claims 26 and 86-99 under 35 U.S.C. 103. Claim 100 depends on claim 26. Winther does not explicitly teach the limitations of claim 100.
Grabinski discloses in situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across numerous research fields. Typically, a research group will put forth significant effort to design, generate, validate and then utilize in situ probes in thin or ultrathin paraffin embedded tissue sections. While combining ISH and IHC is an established technique, the combination of RNAscope ISH, a commercially available ISH assay with single transcript sensitivity, and IHC in thick free-floating tissue sections has not been described. Here, we provide a protocol that combines RNAscope ISH with IHC in thick free-floating tissue sections from the brain and allows simultaneous co-localization of genes and proteins in individual cells. This approach works well with a number of ISH probes (e.g. small proline-rich repeat 1a, βIII-tubulin, tau, and β-actin) and IHC antibody stains (e.g. tyrosine hydroxylase, βIII-tubulin, NeuN, and glial fibrillary acidic protein) in rat brain sections. In addition, we provide examples of combining ISH-IHC dual staining in primary neuron cultures and double-ISH labeling in thick free-floating tissue sections from the brain. Finally, we highlight the ability of RNAscope to detect ectopic DNA in neurons transduced with viral vectors. RNAscope ISH is a commercially available technology that utilizes a branched or “tree” in situ method to obtain ultrasensitive, single transcript detection. Immunohistochemistry is a tried and true method for identifying specific protein in cell populations. The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.
Regarding claim 100, Grabinski teaches a method wherein “The combination of mRNA analysis via ISH and protein analysis via IHC in the same section is an extremely powerful technique that allows one to answer important biological questions that are difficult to address using other methods. Here, we describe a method for combining ISH (i.e. RNAscope technology) with IHC in thick free-floating tissue sections from the brain. This method of ISH is exceptionally sensitive (i.e. single transcript sensitivity) and provides exquisite cellular localization” (Pg. 2, Introduction last para.). “providing cellular localization” is interpreted as mapping spatial organization. Thus, Grabinski teaches a method wherein the method is used for mapping spatial organization in a complex tissue, and optionally wherein the complex tissue is a tumor tissue.
Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of simultaneously detecting a target nucleic acid and a target protein in a biological sample as taught by Winther to incorporate the method for use in mapping spatial organization as suggested by Grabinski and provide method used for mapping spatial organization in a complex tissue sample. Doing so would improve the techniques used to localize and map gene expression within the cell’s architecture.
Response to Arguments
Applicant's arguments filed 10/14/2025 do not apply to the new grounds of rejections. Please see the response to the argument documented above in the rejection of claim 26 under 35 U.S.C. 103.
Claims 26 and 101 is rejected under 35 U.S.C. 103 as being unpatentable over Lars Winther (“Winther”; US Patent App. Pub. US 2010/0105145 A1, April 29, 2010) in view of Kamme et al. (“Kamme”; US Patent App. Pub. No. US 2004/0259133 A1, Dec. 23, 2004).
The teachings of Winther are documented above in the rejection of claims 26 and 86-99 under 35 U.S.C. 103. Claim 101 depends on claim 26.
Regarding claim 101, Winther also teaches a method wherein it is compatible with many known detection formats and their associated samples… All of those detection assays are useful in research as well as in the detection and diagnosis of a variety of diseases and conditions, for example IHC and ISH Detection Systems (Para. 90).
Winther does not explicitly teach the limitations of claim 101.
Kamme discloses methods of analyzing nucleic acid expression patterns involving the preservation of RNA in biological samples in histochemical assays(Abstract).
Regarding claim 101, Kamme teaches a method wherein “histochemically staining the RNA-preserved biological sample; … and analyzing mRNA expression patterns … by a method comprising in-situ hybridization or isolating identified cells and subjecting the isolated cells to bioarray gene profiling. In a preferred embodiment, the histochemically analyzing comprises subjecting the biological sample to a histochemical assay selected from: in situ hybridization for detecting mRNA; fluorescence in-situ hybridization for detecting DNA; immunocytochemistry assay for detecting proteins;” (Para. 9). Kamme teaches a method wherein “gene expression analysis may find use in various applications, e.g., identifying expression of genes, correlating gene expression to a particular phenotype, screening for disease predisposition” Thus, Kamme suggests a method wherein the method is used for detecting altered gene expression in the biological samples from a diseased model.
Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of simultaneously detecting a target nucleic acid and a target protein in a biological sample as taught by Winther to incorporate the method of analyzing expression patterns to identify disease as suggested by Kamme and provide detecting altered gene expression in the biological samples from a diseased model (Col. 18, lines 1-12). Doing so would enhance the identification, characterization, and diagnosis of disease.
Response to Arguments
Applicant's arguments filed 10/14/2025 do not apply to the new grounds of rejections. Please see the response to the argument documented above in the rejection of claim 26 under 35 U.S.C. 103.
Claims 26 and 102 is rejected under 35 U.S.C. 103 as being unpatentable over Lars Winther (“Winther”; US Patent App. Pub. US 2010/0105145 A1, April 29, 2010) in view of Hsi E. D. (2001). A practical approach for evaluating new antibodies in the clinical immunohistochemistry laboratory. Archives of pathology & laboratory medicine, 125(2), 289–294.
The teachings of Winther are documented above in the rejection of claims 26 and 86-99 under 35 U.S.C. 103. Claim 102 depends on claim 26. Winther does not explicitly teach the limitations of claim 102.
Hsi discloses Paraffin section immunohistochemistry (IHC) is widely used in diagnostic surgical pathology. Today, it is difficult to imagine the practice of surgical pathology without IHC. The availability of automated immunostainers with reagent rental contracts makes this technology commonplace. One potential danger is that many laboratories are now offering immunostains without significant prior knowledge or experience in IHC. As part of its mission, the Cell Markers Committee of the College of American Pathologists offers this manuscript as a basic guide to introducing new antibodies in the clinical IHC laboratory. Issues relating to regulatory developments, antibody selection, staining optimization, and test validation are addressed. (Abstract)
Regarding claim 102, Hsi teaches a method wherein “developments in diagnostic IHC, with an emphasis on recommendations for testing new antibodies” (Pg. 293, Summary, Para.1). Thus, His teaches a method wherein the method is used for validating novel antibodies.
Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of simultaneously detecting a target nucleic acid and a target protein in a biological sample as taught by Winther to incorporate the method of validating an antibody as taught by Hsi and provide validating a novel antibody in biological samples. Doing so would improve the method of validating a novel antibody.
Response to Arguments
Applicant's arguments filed 10/14/2025 do not apply to the new grounds of rejections. Please see the response to the argument documented above in the rejection of claim 26 under 35 U.S.C. 103.
Conclusion of Response to Arguments
In view of the amendments, documented above in this Final Office Action are new grounds of rejections as well as responses to arguments. No claims are in condition for allowance.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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 nonprovisional extension fee (37 CFR 1.17(a)) 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Winston Shen can be reached at (571)272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682