DETAILED ACTION
Notice of Pre-AIA or AIA Status
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
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 02/24/2026 has been entered.
Claims 3, 13, 14, and 17-20 were amended in the claim set filed 02/24/2026.
Claims 21-24 were added in the claim set filed 02/24/2026.
Accordingly, claims 3, 13, 14, and 17-20 are pending and under consideration.
Priority
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. The earliest effective filing date to which the instant application is entitled is 05/07/2019.
Status of Prior Objections/Rejections
RE: Claim Rejections - 35 USC § 112(a)
►Claim 18 was previously rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement.
The amendment to instant claim 18 has obviated the basis of the prior rejection. The rejection of record is hereby withdrawn.
RE: Claim Rejections - 35 USC § 112(b)
►Claim 17 was previously rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The amendment to instant claim 17 has obviated the basis of the prior rejection. The rejection of record is hereby withdrawn.
RE: Claim Rejections - 35 USC § 103
►Claims 3, 7, 10, 11, and 12 were previously rejected under 35 U.S.C. 103 as being unpatentable over Lukman et al., 2013 (of record) in view of Diagnostic Automation/Cortez Diagnostics, Inc., 2012 (of record), and Krusiński et al., 2010, as evidenced by Slater and Beato, 1993 (of record).
Applicant has traversed the rejection of record, asserting that Krusiński does not disclose a construct in which FRET occurs between a fluorophore/quencher pair positioned on opposing sides of the hormone response element, as in amended instant claim 3. Applicant further asserts that Krusiński emphasizes the importance of spacing between the fluorophore/quencher pairs taught therein to achieve optimal FRET and therefore, there is no basis in Krusiński to predict that a single fluorophore/quencher pair positioned on opposing sides of the hormone response element would function effectively in a FRET-based assay, as the hormone response element would create a separation that exceeds the effective FRET distance reported by Krusiński as important. Finally, Applicant asserts that modifying the teachings of Lukman as proposed by the Office would alter the fundamental operating principle of Lukman.
In response, Applicant’s assertion that the cited art does not disclose a construct as claimed at amended instant claim 3 is found persuasive. Additionally, Applicant’s assertion regarding modifying the teachings of Lukman is also found persuasive. However, new grounds of rejection are set forth below.
►Claim 5 was previously rejected under 35 U.S.C. 103 as being unpatentable over Lukman et al., 2013 (of record) in view of Diagnostic Automation/Cortez Diagnostics, Inc., 2012 (of record), and Krusiński et al., 2010, as evidenced by Slater and Beato, 1993 (of record), as applied to claim 3 above, and further in view of Smith and Toft, 2008 (of record) and as evidenced by Echeverria and Picard, 2009 (of record).
As set forth above, Applicant’s assertion that the cited art does not disclose a construct as claimed at amended instant claim 3 (from which instant claim 5 directly depends) is found persuasive. However, new grounds of rejection are set forth below.
►Claim 6 was previously rejected under 35 U.S.C. 103 as being unpatentable over Lukman et al., 2013 (of record) in view of Diagnostic Automation/Cortez Diagnostics, Inc., 2012 (of record), and Krusiński et al., 2010, as evidenced by Slater and Beato, 1993 (of record), as applied to claim 3 above and further in view of Smith and Toft, 2008 (of record), as evidenced by Echeverria and Picard, 2009 (of record) as applied to claim 5 above, and further in view of Kang et al., 1999 (of record), as evidenced by Lukman et al., 2013 (of record).
As set forth above, Applicant’s assertion that the cited art does not disclose a construct as claimed at amended instant claim 3 (from which instant claim 6 indirectly depends) is found persuasive. However, new grounds of rejection are set forth below.
►Claims 13, 19, and 20 were previously rejected under 35 U.S.C. 103 as being unpatentable over Lukman et al., 2013 (of record), in view of Krusiński et al., 2010, and Smith and Toft, 2008 (of record), as evidenced by Slater and Beato, 1993 (of record), and Echeverria and Picard, 2009 (of record).
While claim 13 does not depend from instant claim 3, the amended subject matter recited therein is identical to that of amended instant claim 3. This is consistent with Applicant’s assertion that the cited art fails to render the claimed subject matter obvious for at least the reasons set forth above with respect to instant claim 3.
As set forth above, Applicant’s assertion that the cited art does not disclose a construct as claimed at amended instant claim 13 is found persuasive. However, new grounds of rejection are set forth below.
►Claim 14 was previously rejected under 35 U.S.C. 103 as being unpatentable over Lukman et al., 2013 (of record) in view of Krusiński et al., 2010, and Smith and Toft, 2008 (of record), as evidenced by Slater and Beato, 1993 (of record), and Echeverria and Picard, 2009 (of record) as applied to claim 13 above, and further in view of Anawalt, 2017 (of record).
As set forth above, Applicant’s assertion that the cited art does not disclose a construct as claimed at amended instant claim 13 (from which instant claim 14 directly depends) is found persuasive. However, new grounds of rejection are set forth below.
►Claim 15 was previously rejected under 35 U.S.C. 103 as being unpatentable over Lukman et al., 2013 (of record) in view of Krusiński et al., 2010, and Smith and Toft, 2008 (of record) as evidenced by Slater and Beato, 1993 (of record), and Echeverria and Picard, 2009 (of record), as applied to claim 13 above and further in view of Anawalt, 2017 (of record) as applied to claim 14 above, and further in view of the Equine Anti-Doping and Controlled Medication Regulations of the International Federation for Equestrian Sports, 2018 (of record).
As set forth above, Applicant’s assertion that the cited art does not disclose a construct as claimed at amended instant claim 13 (from which instant claim 15 indirectly depends) is found persuasive. However, new grounds of rejection are set forth below.
►Claim 17 was previously rejected under 35 U.S.C. 103 as being unpatentable over Lukman et al., 2013 (of record) in view of Krusiński et al., 2010, and Smith and Toft, 2008 (of record), as evidenced by Slater and Beato, 1993 (of record), and Echeverria and Picard, 2009 (of record) as applied to claim 13 above, and further in view of Liu and Lilley, 2017.
As set forth above, Applicant’s assertion that the cited art does not disclose a construct as claimed at amended instant claim 3 (from which instant claim 17 directly depends) is found persuasive. However, new grounds of rejection are set forth below.
New/Maintained Grounds of Objection/Rejection
Claim Objections
Claims 12 and 13 are objected to because of the following informalities:
With regard to claim 12, the recitation of “SEQ ID Nos:” at embodiments (i)-(v) of the claimed test kit, while not strictly improper, is nonetheless inconsistent with standard conventions that recite “SEQ ID NOs:” (bolded and underlined emphasis added). For purposes of comporting with standard conventions, it would be remedial to recite “SEQ ID NOs:” (bolded and underlined emphasis added). Furthermore, embodiment (iii) of the claimed test kit does not comport with standard grammatical and/or linguistic conventions, as there is no article such as “a” preceding the claimed “progesterone receptor.” It would be remedial to amend the instant claim language to recite “a progesterone receptor” in order to comport with standard grammatical and/or linguistic conventions.
With further regard to claim 12, which recites “the test kit according to claim 3, wherein the: (i) the…(ii) the… (iii) the… (iv) the… or (v)… the…” (bolded and underlined emphasis added), which does not comport with standard grammatical and/or linguistic conventions. The double recitation of “the” is not grammatically proper. It would be remedial to amend the instant claim language to comport with standard grammatical and/or linguistic conventions, for example by eliminating the article preceding the recited test kit configuration options or by eliminating the articles preceding the recited configuration of each claimed test kit.
With regard to claim 13, the recitation of step (c), component 3 does not comport with standard grammatical and/or linguistic conventions. A punctuation mark such as a comma or a semicolon is required after the recitation of component 3 prior to continuing to recite the claimed method. It would be remedial to amend the instant claim language to comport with standard grammatical and/or linguistic conventions, for example by inserting a punctuation mark such as a comma or semicolon after the recitation of component 3, as set forth above.
Claim 13 is further objected to for including periods in the recitation of the components of the claimed double stranded nucleic acid reporter construct (c). According to MPEP 608.01(m), “Each claim begins with a capital letter and ends with a period. Periods may not be used elsewhere in the claims except for abbreviations. See Fressola v. Manbeck, 36 USPQ2d 1211 (D.D.C. 1995)”. It would be remedial to replace the periods of instant claim 13 with parentheses (i.e. (1), (2), and (3)) as in the rest of the instant claim set.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 12 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 12 is drawn to a test kit for screening a sample for the presence of a ligand capable of eliciting a steroid hormone genomic response, said test kit comprising a double stranded nucleic acid reporter construct, itself being selected from any one of SEQ ID NOs: 1-70 (for a ligand that binds to an androgen receptor), SEQ ID NOs: 71-100 (for a ligand that binds to an estrogen receptor), SEQ ID NOs: 101-130 (for a ligand that binds to a progesterone receptor), SEQ ID NOs: 131-160 (for a ligand that binds to a mineralocorticoid receptor), or SEQ ID NOs: 131-160 (for a ligand that binds to a glucocorticoid receptor). The claimed double stranded nucleic acid reporter construct is recited to comprise a hormone response element that is capable of being bound by the receptor-ligand complex, a fluorescence generating moiety, and a fluorescence quenching moiety. The rejected claim thus comprises a set of double stranded nucleic acid reporter constructs comprising a hormone response element that is capable of being bound by the receptor-ligand complex, a fluorescence generating moiety, and a fluorescence quenching moiety, wherein said reporter constructs are selected from SEQ ID NOs: 1-160, which are of variable lengths ranging from 15 bp to 30 bp.
In the instant case, sequence searches indicate that at least SEQ ID NO: 1 corresponds solely to the androgen response element (WO 2005/073393 A1; hereinafter Hamilton; Table 3-SEQ ID NO: 41). This androgen response element is only one of the three components of the recited double stranded nucleic acid reporter set forth above. The sequence listing does not require any fluorescence generating moiety or any fluorescence quenching moiety. It is thus unclear how SEQ ID NO: 1 could correspond to the claimed reporter construct, as recited at instant claim 11, as SEQ ID NO: 1 does not include all three components of the recited double stranded nucleic acid reporter set forth above.
To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof. The specification describes reporter constructs comprising fluorophores including fluorescent donors (Cy3) and acceptors (Cy5) at Examples 1 and 2. No description is provided of a double stranded nucleic acid reporter construct that does not include fluorescent donors and acceptors.
Even if one accepts that the examples described in the specification meet the claim limitations of the rejected claims with regard to structure and function, the examples are only representative of double stranded nucleic acid reporter construct comprising fluorescent donors and acceptors in addition to a steroid hormone response element. The results are not necessarily predictive of double stranded nucleic acid reporter constructs comprising only a steroid hormone response element, as encompassed by the instant claim set. Thus, it is impossible for one to extrapolate from the few examples described herein those assays (and the test kits comprising them) that would necessarily meet the structural/functional characteristics of the rejected claims.
The prior art does not appear to offset the deficiencies of the instant specification in that it does not describe a set of test kits comprising assays to detect ligands capable of forming a receptor-ligand complex with a steroid hormone receptor, said assays comprising double stranded nucleic acid reporter constructs comprising only a steroid hormone response element, as encompassed by the instant claim set.
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claim 12.
Claims 21 and 22 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 21 and 22 are drawn to an assay method for screening a sample for the presence of a ligand capable of eliciting a steroid hormone genomic response, said test kit comprising a Cy5 fluorescence generating moiety and a Cy3 fluorescence quenching moiety. The rejected claim thus comprises an assay in which Cy5 generates a fluorescent signal and Cy3 quenches the fluorescent signal readout of said assay.
To provide adequate written description and evidence of possession of a claimed invention, the specification must provide sufficient distinguishing identifying characteristics, such as by including disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof. In the instant case, while the specification describes an assay for screening a sample for the presence of a ligand capable of eliciting a steroid hormone genomic response, said assay comprising a Cy3 fluorescence generating moiety and a Cy5 fluorescence quenching moiety (Example 1 - pages 69-74), no description is provided of an assay comprising Cy3 as a fluorescence quenching moiety or of Cy5 as a fluorescence generating moiety, as instantly claimed.
The prior art does not appear to offset the deficiencies of the instant specification in that it does not describe an assay in which Cy3 functions as a fluorescence quenching moiety. The prior art teaches that Cy3 is a commonly-used fluorescence generating moiety that is also commonly paired with the fluorescence-quenching moiety Cy5 (i.e. a donor-acceptor pair of Cy3-Cy5) (Liu and Lilley, 2017-abstract). This is consistent with the Examples provided in the instant specification (see Example 1).
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 21 and 22, as such an invention was not disclosed in the instant specification, nor has it been disclosed in the prior art.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 14 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). Instant claim 14 recites both that “an increase in fluorescence of the reporter construct” indicates the presence of a “ligand sufficient to activate a steroid hormone receptor” in a sample obtained from an athlete and that “a change in fluorescence of the reporter construct provides information about the doping status of the athlete.” However, an increase is only one possible type of change in fluorescence of the reporter construct. While a decrease may also be used as a readout of ligand presence (see Example 1, Section 1.1 of the instant specification), the instant claim is explicitly drawn to increasing fluorescent reporter signal. Furthermore, one of ordinary skill in the art would reasonably conclude that the absence of a ligand will not change the fluorescent reporter signal, as no binding would occur to alter the energy transfer between the fluorescence generating moiety and the fluorescence quenching moiety. Therefore, lack of change in fluorescence of the reporter construct would also be reasonably considered to provide information about the doping status of the tested athlete. As currently constructed, the instant claim is not clear regarding the metes and bounds of protection sought, as it is unclear whether solely an increase in fluorescence of the reporter construct provides information about the doping status of the tested athlete or whether any change in fluorescence of the reporter construct provides information about the doping status of the tested athlete. It would be remedial to amend the instant claim language such that one of ordinary skill in the art would be reasonably apprised of the bounds of protection sought, for example in the instant case whether the claimed assay readout is informed by an increase in fluorescence of the reporter construct or any change to the same.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 3, 7, 10, 11, and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Cooper., 2013 (hereinafter Cooper; as cited in the IDS filed 03/22/2022) in view of Krusiński et al., 2010 (hereinafter Krusiński; of record), US 2006/0112440 A1 (hereinafter Tsien), and Automation/Cortez Diagnostics, Inc., 2012 (hereinafter Automation; of record), as evidenced by Slater and Beato, 1993 (hereinafter Slater; of record), Guével and Pakdel, 2001 (hereinafter Guével), and WO 2005/073396 A1 (hereinafter Hamilton).
With regard to amended claim 3, which recites “a test kit for screening a sample for the presence of a ligand capable of eliciting a steroid hormone genomic response…comprising: (i) a steroid hormone receptor…and (ii) a double stranded nucleic acid reporter construct comprising: (a) a hormone response element that is capable of being bound by the receptor-ligand complex; and (b) a fluorescence generating moiety; and (c) a fluorescence quenching moiety wherein the hormone response element (a) is located between the fluorescence generating moiety (b) and the fluorescence quenching moiety (c), and wherein, the fluorescence generating moiety (b) and the fluorescence quenching moiety (c) are positioned on opposing sides of the hormone response element, and wherein fluorescence quenching occurs between the fluorescence generating moiety (b) and the fluorescence quenching moiety (c) in the absence of the receptor-ligand complex,” Cooper discloses that in vitro bioassays to detect steroids such as androgens are of increasing importance in combating androgen doping associated with nutritional supplements taken illicitly to enhance sporting performance and/or physical appearance (Abstract). These assays are of increasing importance, as many designer androgens cannot be detected by standard highly-sensitive screening methods due to their chemical structures that differ from known androgen structures (Abstract; Section 1. Introduction). There is therefore a need for a suitable tool, such as in vitro androgen bioassays, for routine screening of nutraceutical or biological samples suspected to contain an androgen (Section 1. Introduction).
Such in vitro bioassays include the methods taught in Krusiński, which discloses a double-stranded nucleic acid reporter construct comprising a fluorescence generating moiety and a fluorescence quenching moiety flanking a hormone response element for detecting binding of ecdysteroid receptor DNA binding domains to the associated response element by observing fluorescence signal quenching as a result of the resonance energy transfer between the donors and quenchers (Figure 1; Abstract; Page 2, Column 1, Paragraph 4-Page 2, Column 2, Paragraph 1). As is known to those of ordinary skill in the art, hormone response elements only bind ligand-receptor complexes (Slater: Paragraph 1). Krusiński et al., 2010 further discloses that the methods taught therein are inexpensive to practice and generate quality results that are consistent with those obtained by conventional fluorescence titrations and by FRET measurement (page 9, column 1, paragraph 2).
Thus, one of ordinary skill in the art would consider that Krusiński discloses an in vitro assay for detecting a ligand capable of eliciting a steroid hormone genomic response, wherein said assay comprises contacting a double stranded nucleic acid reporter construct comprising a hormone response element capable of being bound by the assayed steroid hormone receptor-ligand complex (i.e. ecdysteroid receptor proteins ultraspiracle and ecdysone receptor) and flanked by a fluorescence generating moiety and a fluorescence quenching moiety with said steroid hormone receptor-ligand complex (Abstract; Figure 1). However, Krusiński does not disclose a construct in which FRET occurs between a fluorophore/quencher pair positioned on opposing sides of the hormone response element, as set forth by Applicant in the arguments filed 02/24/2026.
This deficiency is cured by Tsien. Tsien discloses fluorescent protein sensors for detection of analytes, wherein a binding protein moiety (written as “building protein moiety” in Figure 1 but corrected to “binding protein moiety” throughout the rest of the disclosure-see Paragraphs [0006] and [0012]) is flanked by a donor fluorescent protein moiety (i.e. a fluorescence generating moiety) and an acceptor fluorescent moiety (i.e. a fluorescence quenching moiety) (Abstract; Figure 1; Paragraph [0006]). This interpretation of donor and acceptor fluorescent moieties is supported by the substitute specification filed 06/03/2022 at page 44, lines 10-19. Tsien specifically discloses that the binding protein moiety taught therein may be a steroid hormone receptor or a ligand binding domain of a steroid hormone receptor and has an analyte-binding region which binds an analyte (i.e. the steroid associated with the steroid hormone receptor set forth above) and causes the fluorescent indicator to change conformation upon exposure to the analyte, thereby altering the fluorescent readout of the system (Paragraphs [0006], [0012], and [0053]-[0054]). The acceptor fluorescent moiety of Tsien is disclosed to be indirectly linked to the binding protein moiety via a linker moiety of variable length (Figure 1; Paragraph [0011]). Finally, Tsien discloses that the systems taught therein may be encoded by nucleic acids (Paragraphs [0019], [0076], and [0096]).
Thus, Tsien discloses fluorescent sensor assays, wherein the fluorescence generating moiety and the fluorescence quenching moiety flank a binding protein moiety, and wherein upon binding of the analyte to said binding protein moiety, the fluorescent protein moieties come close together or physically separate, thereby increasing or decreasing FRET readout (Figure 1: paragraph [0054]).
While none of the cited art individually discloses the instantly claimed test kit, the cited art does teach the components of the instantly claimed test kit, such that it would have been obvious to someone of ordinary skill in the art prior to the effective filing date of the claimed invention to arrive at the instantly claimed invention. First, Cooper teaches that in vitro androgen bioassays are of increasing importance for screening of nutraceutical or biological samples suspected to contain an androgen, as set forth above. While Krusiński discloses such assays comprising a hormone response element flanked by a fluorescence generating moiety and a fluorescence quenching moiety (set forth above), as set forth by Applicant in the arguments filed 02/24/2026, the separation distance between the fluorescent moieties taught therein is crucial to achieve optimal FRET and is not reasonably predicted to extrapolate to a single fluorophore/quencher pair, as the requisite separation would exceed the effective FRET distance reported by Krusiński as important. However, Tsien discloses FRET between a single fluorophore/quencher pair, wherein said fluorophore/quencher pair is separated by a binding protein moiety (such as a steroid hormone receptor or a ligand binding domain of a steroid hormone receptor) with an analyte-binding region which causes the fluorescent indicator to change conformation upon binding of the analyte (i.e. he steroid associated with the steroid hormone receptor set forth above), thereby altering the fluorescent readout of the system. Crucially, the distance between the fluorophore/quencher pair is disclosed to be tunable by varying the length of the linker indirectly connecting the binding protein moiety and the acceptor fluorescent moiety. Therefore, it would have been obvious to someone of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the assay disclosed in Krusiński to overcome the limitations regarding the distance of separation between the fluorescent moieties to achieve optimal FRET based on the disclosure of Tsien, which teaches a single fluorophore/quencher pair with tunable separation distance; for example, by substituting the hormone response element of Krusiński for the binding protein moiety of Tsien such that the double stranded nucleic acid reporter construct of Krusiński is flanked by a single fluorophore/quencher pair with tunable separation distance to optimize FRET (as disclosed in Tsien) in order to predictably detect analytes (such as steroids) in the tested sample (as motivated by Cooper) by detecting increased or decreased FRET readout, (as disclosed in Krusiński and Tsien). One would have been motivated to make such a modification in order to receive the expected benefit of producing an assay capable of predictably and inexpensively detecting steroid hormones via FRET readout produced by their interactions with their associated response elements.
Additionally, while none of the cited art discloses the packaging of these components into a test kit, such test kits to detect steroid ligands were publicly available prior to the effective filing date of the claimed invention. Using the Wayback Machine, Automation sold steroid test kits as early as if not earlier than September 22, 2012. Thus, Automation establishes that standardized test kits for detecting steroid hormone, albeit ones that detect steroid hormones via different methods than instantly claimed, were on sale and available to the public prior to the effective filing date of the claimed invention. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to package the assay set forth above into a standardized kit, as demonstrated by Automation. One would have been motivated to make such a modification in order to receive the expected benefit of producing an inexpensive and effective test kit for detecting ligands capable of eliciting a steroid hormone genomic response.
With regard to claim 7, which recites “the relative amount of steroid hormone receptor to nucleic acid reporter construct is y:1, where y is the amount of steroid hormone receptor and is defined as [1.0 < y < 5.0],” Krusiński discloses experimentation with both nucleic acid reporter construct concentration and protein concentration (i.e. ecdysteroid receptor proteins ultraspiracle (Usp) and ecdysone receptor (EcR)) (Figures 2 and 4; Page 5, Column 2, Paragraph 2-Page 8, Column 1, Paragraph 1). Thus, based on the disclosure of Krusiński, experimentation with the concentration of assay components for optimization thereof constitutes routine experimentation. Per MPEP 2144.05.I.A, “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)). Thus, it is considered that Krusiński discloses each and every additional limitation of instant claim 7.
With regard to claim 10, which recites “the steroid hormone receptor is selected from the group consisting of…estrogen receptor alpha (ER-α) [and] estrogen receptor beta (ER-β),” as discussed regarding instant claim 3, Cooper specifically discloses that in vitro bioassays to detect steroids such as androgens are of increasing importance in combating androgen doping associated with nutritional supplements taken illicitly to enhance sporting performance and/or physical appearance (Abstract). There is therefore a need for a suitable tool, such as in vitro androgen bioassays, for routine screening of nutraceutical or biological samples suspected to contain an androgen (Section 1. Introduction). Thus, while none of the cited art teaches the instantly claimed assay for detecting androgens, one of ordinary skill in the art would have been motivated by the disclosure of Cooper prior to the effective filing date of the claimed invention to modify the assay of the test kit set forth above (regarding instant claim 3) such that the assayed steroid receptor is specifically an androgen receptor for purposes of detecting illicit androgen doping. Thus, it is considered that Cooper discloses each and every additional limitation of instant claim 10.
With regard to claim 11, which recites “the steroid hormone receptor is…a recombinant steroid hormone receptor,” as discussed regarding instant claim 3, Cooper specifically discloses that in vitro bioassays to detect steroids such as androgens are of increasing importance in combating androgen doping associated with nutritional supplements taken illicitly to enhance sporting performance and/or physical appearance (Abstract). Such in vitro bioassays include cell-based androgen bioassays, wherein the queried androgen receptor is supplied to yeast in recombinant form via a plasmid vector encoding the androgen receptor (not expressed in yeast cells) and β-galactosidase (Figure 2). The recombinant nature of the supplied androgen receptor is reviewed in Guével, which reports the production of recombinant yeast expressing the estrogen receptor, wherein said recombinant yeast are rapidly and accurately identified by assaying for the exogenously-supplied β-galactosidase (see Abstract in particular). The explicitly recombinant estrogen receptor reported in Guével supports the conclusion that the androgen receptor reported in Figure 2 (specifically the yeast in vitro cell assay) of Cooper is also a recombinant androgen receptor. Thus, it is considered that Cooper, as evidenced by Guével, discloses each and every additional limitation of instant claim 11.
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With regard to claim 12, which recites “(i) the test kit [of claim 3] is configured to detect a ligand that binds to an androgen receptor and the reporter construct is selected from any one of SEQ ID Nos: 1-70,” as discussed regarding instant claim 3, Cooper specifically discloses that in vitro bioassays to detect steroids such as androgens are of increasing importance in combating androgen doping associated with nutritional supplements taken illicitly to enhance sporting performance and/or physical appearance (Abstract). While Cooper discloses in vitro assays for detecting steroids such as androgens (see Figure 2), Cooper is silent as to the sequence of the androgen response element utilized therein. However, this sequence is a standard sequence that has been known in the field, as evidenced by Hamilton, which discloses that SEQ ID NO: 41 taught therein corresponds to the androgen response element (Table 3). As shown in the alignment below, SEQ ID NO: 41 of Hamilton is 100% identical to instant SEQ ID NO: 1, as recited at instant claim 12.
Thus, it is considered that Cooper, as evidenced by Hamilton, discloses each and every additional limitation of instant claim 12.
Given that Cooper, Krusiński, Tsien and Automation (as evidenced by Slater, Guével, and Hamilton) collectively disclose the test kit of instant claim 3, as set forth above, and that Krusiński and Cooper (as evidenced by Guével and Hamilton) disclose the additional limitations of instant claims 7, 10, 11, and 12, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to optimize the ratio of nucleic acid reporter construct to steroid hormone receptor (as disclosed in Krusiński), wherein the ligand of interest specifically corresponds to the recombinant androgen receptors taught in Cooper and set forth above, to predictably detect analytes (such as steroids) in the tested sample (as motivated by Cooper) by detecting increased or decreased FRET readout, (as disclosed in Krusiński and Tsien). One would have been motivated to make such a modification in order to receive the expected benefit of producing an inexpensive and effective test kit (as disclosed by Automation) for detecting ligands capable of eliciting a steroid hormone genomic response.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Cooper., 2013 (hereinafter Cooper; as cited in the IDS filed 03/22/2022) in view of Krusiński et al., 2010 (hereinafter Krusiński; of record), US 2006/0112440 A1 (hereinafter Tsien), and Automation/Cortez Diagnostics, Inc., 2012 (hereinafter Automation; of record), as evidenced by Slater and Beato, 1993 (hereinafter Slater; of record), Guével and Pakdel, 2000 (hereinafter Guével), and WO 2005/073396 A1 (hereinafter Hamilton), as applied to claim 3 above, and further in view of Smith and Toft, 2008 (hereinafter Smith; of record), as evidenced by Echeverria and Picard, 2009 (hereinafter Echeverria; of record).
The combined disclosures of Cooper, Krusiński, Tsien, Automation, Slater, Guével, and Hamilton are described above and applied as before. However, these disclosures do not teach the steroid hormone receptor cofactor of instant claim 5.
With regard to claim 5, which recites “the test kit [of claim 3]…further compris[es] a steroid hormone receptor cofactor selected from heat shock protein 90 (HSP90),” as well as various complexes of HSP90, HSP70, HSP40, p23, Hop, Hip, p60, and/or FKBP52, Smith teaches that heat shock proteins in steroid receptor complexes (i.e. Hsp70, Hsp40, and Hsp90), along with cochaperones (i.e. Hsp90-associated cochaperones) are required both to establish steroid receptor binding ability, as well as to maintain its binding ability (Page 2229-Paragraph 1; Section "Hormone Binding Ability and Chaperones"-Paragraph 1). Additionally, Echeverria depicts Hsp90 machinery associated with nuclear localization, which includes the species recited in instant claim 5 (i.e. Hsp90, Hsp70, p23, HOP, and Hsp40) in addition to other cofactors (Figure 1).
Given that Cooper, Krusiński, Tsien and Automation (as evidenced by Slater, Guével, and Hamilton) collectively disclose the test kit of instant claim 3, as set forth above, and that hormone receptor cofactors such as HSP90 are crucial for facilitating and maintaining steroid hormone-receptor binding, as taught by Smith and as evidenced by Echeverria, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to apply the teachings of Smith to the test kit of instant claim 3 to predictably promote and enhance receptor-ligand formation and persistence to more accurately detect the ligand of interest. One would have been motivated to make such an addition to the test kit in order to receive the expected benefit of increased detection accuracy.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Cooper., 2013 (hereinafter Cooper; as cited in the IDS filed 03/22/2022) in view of Krusiński et al., 2010 (hereinafter Krusiński; of record), US 2006/0112440 A1 (hereinafter Tsien), and Automation/Cortez Diagnostics, Inc., 2012 (hereinafter Automation; of record), as evidenced by Slater and Beato, 1993 (hereinafter Slater; of record), Guével and Pakdel, 2000 (hereinafter Guével), and WO 2005/073396 A1 (hereinafter Hamilton), and further in view of Smith and Toft, 2008 (hereinafter Smith; of record), as evidenced by Echeverria and Picard, 2009 (hereinafter Echeverria; of record) as applied to claim 5 above, and further in view of Kang et al., 1999 (hereinafter Kang; of record).
The combined disclosures of Cooper, Krusiński, Tsien, Automation, Slater, Guével, Hamilton, Smith, and Ecehverria are described above and applied as before. However, these disclosures do not teach the HSP90 ratio of instant claim 6.
With regard to claim 6, which recites “the relative amount of HSP90 to steroid hormone receptor is x:1, where x is the amount of HSP90 and is defined as [1.0 < x < 5.0],” Kang teaches that the ratio of Hsp90 to glucocorticosteroid receptor impacts both receptor-response element binding as well as ligand responsiveness (abstract). As set forth above regarding instant claim 5, cofactors such as HSP90 are required to facilitate and maintain hormone-receptor binding, as taught by Smith, and also to govern receptor-response element binding and ligand responsiveness, as taught by Kang.
While Kang does not teach the range of ratios disclosed in instant claim 6, they do teach that these ratios impact receptor-ligand interactions, which in turn impact the accuracy of ligand detection using the instantly claimed test kit. Per MPEP 2144.05.II.A, “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)). Thus, given the teachings of Kang regarding the importance of HSP90 to glucocorticosteroid receptor ratio, it would have been obvious to someone of ordinary skill in the art before the filing date of the claimed invention to establish optimum or workable ranges by routine experimentation.
Given that the test kit recited at instant claim 3 (and collectively disclosed by Cooper, Krusiński, Tsien and Automation, as evidenced by Slater, Guével, and Hamilton) relies upon binding of a receptor-ligand complex to a hormone response element, which is facilitated by hormone receptor cofactors such as HSP90 (set forth above regarding instant claim 5), and that the ratio of these hormone receptor cofactors to receptors such as the glucocorticosteroid receptor impacts both receptor-response element binding and ligand responsiveness, as taught by Kang, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to apply the teachings of Kang to practice routine experimentation to establish an optimum or workable range to predictably promote receptor-response element binding as well as ligand responsiveness to more accurately detect the ligand of interest. One would have been motivated to make such an addition to the test kit in order to receive the expected benefit of increased detection accuracy.
Claims 13-15, 19, 20, 23, and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Cooper., 2013 (hereinafter Cooper; as cited in the IDS filed 03/22/2022) in view of Krusiński et al., 2010 (hereinafter Krusiński; of record), US 2006/0112440 A1 (hereinafter Tsien), Smith and Toft, 2008 (hereinafter Smith; of record), and Kang et al., 1999 (hereinafter Kang; of record), as evidenced by Slater and Beato, 1993 (hereinafter Slater; of record) and Echeverria and Picard, 2009 (hereinafter Echeverria; of record).
With regard to amended claim 13, which recites “an assay method for detecting a ligand in a sample, which ligand is capable of eliciting a steroid hormone genomic response, the method comprising the steps of: (i) contacting the sample with: (a) a steroid hormone receptor that forms a receptor-ligand complex with a ligand from the test sample; (b) optionally, heat shock protein 90 (HSP90)” as well as various complexes of HSP90, HSP70, HSP40, p23, Hop, Hip, p60, and/or FKBP52 “and (c) a double stranded nucleic acid reporter construct comprising: 1. a hormone response element that is bound by the receptor-ligand complex; 2. a fluorescence generating moiety; and 3. a fluorescence quenching moiety wherein the hormone response element (1) is located between the fluorescence generating moiety (2) and the fluorescence quenching moiety (3); and wherein, the fluorescence generating moiety (2) and the fluorescence quenching moiety (3) are positioned on opposing sides of the hormone response element, and wherein fluorescence quenching occurs between the fluorescence generating moiety (2) and the fluorescence quenching moiety (3) in the absence of the receptor-ligand complex; and (ii) measuring fluorescence of the reporter construct, wherein, a measured increase in fluorescence of the reporter construct reflects that a ligand has been detected in the sample,” as set forth above regarding instant claim 3, Cooper discloses that in vitro bioassays to detect steroids such as androgens are of increasing importance in combating androgen doping associated with nutritional supplements taken illicitly to enhance sporting performance and/or physical appearance (Abstract). These assays are of increasing importance, as many designer androgens cannot be detected by standard highly-sensitive screening methods due to their chemical structures that differ from known androgen structures (Abstract; Section 1. Introduction). There is therefore a need for a suitable tool, such as in vitro androgen bioassays, for routine screening of nutraceutical or biological samples suspected to contain an androgen (Section 1. Introduction).
Such in vitro bioassays include the methods taught in Krusiński, which discloses a double-stranded nucleic acid reporter construct comprising a fluorescence generating moiety and a fluorescence quenching moiety flanking a hormone response element for detecting binding of ecdysteroid receptor DNA binding domains to the associated response element by observing fluorescence signal quenching as a result of the resonance energy transfer between the donors and quenchers (Figure 1; Abstract; Page 2, Column 1, Paragraph 4-Page 2, Column 2, Paragraph 1). As is known to those of ordinary skill in the art, hormone response elements only bind ligand-receptor complexes (Slater: Paragraph 1). Krusiński et al., 2010 further discloses that the methods taught therein are inexpensive to practice and generate quality results that are consistent with those obtained by conventional fluorescence titrations and by FRET measurement (page 9, column 1, paragraph 2).
Thus, one of ordinary skill in the art would consider that Krusiński discloses an in vitro assay for detecting a ligand capable of eliciting a steroid hormone genomic response, wherein said assay comprises contacting a double stranded nucleic acid reporter construct comprising a hormone response element capable of being bound by the assayed steroid hormone receptor-ligand complex (i.e. ecdysteroid receptor proteins ultraspiracle and ecdysone receptor) and flanked by a fluorescence generating moiety and a fluorescence quenching moiety with said steroid hormone receptor-ligand complex (Abstract; Figure 1). However, Krusiński does not disclose a construct in which FRET occurs between a fluorophore/quencher pair positioned on opposing sides of the hormone response element, as set forth by Applicant in the arguments filed 02/24/2026.
This deficiency is cured by Tsien. Tsien discloses fluorescent protein sensors for detection of analytes, wherein a binding protein moiety (written as “building protein moiety” in Figure 1 but corrected to “binding protein moiety” throughout the rest of the disclosure-see Paragraphs [0006] and [0012]) is flanked by a donor fluorescent protein moiety (i.e. a fluorescence generating moiety) and an acceptor fluorescent moiety (i.e. a fluorescence quenching moiety) (Abstract; Figure 1; Paragraph [0006]). This interpretation of donor and acceptor fluorescent moieties is supported by the substitute specification filed 06/03/2022 at page 44, lines 10-19. Tsien specifically discloses that the binding protein moiety taught therein may be a steroid hormone receptor or a ligand binding domain of a steroid hormone receptor and has an analyte-binding region which binds an analyte (i.e. the steroid associated with the steroid hormone receptor set forth above) and causes the fluorescent indicator to change conformation upon exposure to the analyte, thereby altering the fluorescent readout of the system (Paragraphs [0006], [0012], and [0053]-[0054]). The acceptor fluorescent moiety of Tsien is disclosed to be indirectly linked to the binding protein moiety via a linker moiety of variable length (Figure 1; Paragraph [0011]). Finally, Tsien discloses that the systems taught therein may be encoded by nucleic acids (Paragraphs [0019], [0076], and [0096]).
Thus, Tsien discloses fluorescent sensor assays, wherein the fluorescence generating moiety and the fluorescence quenching moiety flank a binding protein moiety, and wherein upon binding of the analyte to said binding protein moiety, the fluorescent protein moieties come close together or physically separate, thereby increasing or decreasing FRET readout (Figure 1: paragraph [0054]).
Thus, Cooper, Krusiński, and Tsien collectively disclose each and every limitation of amended instant claim 13, with the exception of the optional hormone receptor cofactors. This deficiency is cured by Smith, which teaches that heat shock proteins in steroid receptor complexes (i.e. Hsp70, Hsp40, and Hsp90), along with cochaperones (i.e. Hsp90-associated cochaperones) are required both to establish steroid receptor binding ability, as well as to maintain its binding ability (Page 2229-Paragraph 1; Section "Hormone Binding Ability and Chaperones"-Paragraph 1). In support of this, Echeverria depicts Hsp90 machinery associated with nuclear localization, which includes the species recited in instant claim 5 (i.e. Hsp90, Hsp70, p23, HOP, and Hsp40) in addition to other cofactors (Figure 1).
While none of the cited art individually discloses the instantly claimed method, the cited art does teach the components of the instantly claimed method, such that it would have been obvious to someone of ordinary skill in the art prior to the effective filing date of the claimed invention to arrive at the instantly claimed invention. First, Cooper teaches that in vitro androgen bioassays are of increasing importance for screening of nutraceutical or biological samples suspected to contain an androgen, as set forth above. While Krusiński discloses such assays comprising a hormone response element flanked by a fluorescence generating moiety and a fluorescence quenching moiety (set forth above), as set forth by Applicant in the arguments filed 02/24/2026, the separation distance between the fluorescent moieties taught therein is crucial to achieve optimal FRET and is not reasonably predicted to extrapolate to a single fluorophore/quencher pair, as the requisite separation would exceed the effective FRET distance reported by Krusiński as important. However, Tsien discloses FRET between a single fluorophore/quencher pair, wherein said fluorophore/quencher pair is separated by a binding protein moiety (such as a steroid hormone receptor or a ligand binding domain of a steroid hormone receptor) with an analyte-binding region which causes the fluorescent indicator to change conformation upon binding of the analyte (i.e. he steroid associated with the steroid hormone receptor set forth above), thereby altering the fluorescent readout of the system. Crucially, the distance between the fluorophore/quencher pair is disclosed to be tunable by varying the length of the linker indirectly connecting the binding protein moiety and the acceptor fluorescent moiety. Therefore, it would have been obvious to someone of ordinary skill in the art prior to the effective filing date of the claimed invention to modify the assay disclosed in Krusiński to overcome the limitations regarding the distance of separation between the fluorescent moieties to achieve optimal FRET based on the disclosure of Tsien, which teaches a single fluorophore/quencher pair with tunable separation distance; for example, by substituting the hormone response element of Krusiński for the binding protein moiety of Tsien such that the double stranded nucleic acid reporter construct of Krusiński is flanked by a single fluorophore/quencher pair with tunable separation distance to optimize FRET (as disclosed in Tsien) in order to predictably detect analytes (such as steroids) in the tested sample (as motivated by Cooper) by detecting increased or decreased FRET readout, (as disclosed in Krusiński and Tsien), as facilitated by their cofactors such as Hsp90 (as taught in Smith and Echeverria). One would have been motivated to make such a modification in order to receive the expected benefit of predictably and inexpensively detecting steroid hormones via FRET readout produced by their interactions with their associated response elements as facilitated by their cofactors such as Hsp90 and its associated complexes.
With regard to amended claim 14, which recites “a method for determining the doping status of an athlete, the method comprising performing an assay method according to claim 13 on a sample obtained from the athlete to ascertain if the sample comprises a ligand sufficient to activate a steroid hormone receptor and cause an increase in fluorescence of the reporter construct, wherein a change in fluorescence of the reporter construct provides information about the doping status of the athlete,” as set forth above, Cooper, Krusiński, Tsien, Smith, Slater, and Echeverria collectively disclose the assay method of claim 13. Additionally, as set forth above, Cooper discloses that in vitro bioassays to detect steroids such as androgens are of increasing importance in combating androgen doping associated with nutritional supplements taken illicitly by athletes to enhance sporting performance and/or physical appearance (Abstract; Section 1. Introduction). These assays are of increasing importance, as many designer androgens cannot be detected by standard highly-sensitive screening methods due to their chemical structures that differ from known androgen structures (Abstract; Section 1. Introduction). There is therefore a need for a suitable tool, such as in vitro androgen bioassays, for routine screening of nutraceutical or biological samples from athletes suspected to contain an androgen (Section 1. Introduction). Furthermore, as set forth above, Tsien discloses fluorescent sensor assays, wherein the fluorescence generating moiety and the fluorescence quenching moiety flank a binding protein moiety, and wherein upon binding of the analyte to said binding protein moiety, the fluorescent protein moieties come close together or physically separate, thereby increasing or decreasing FRET readout (Figure 1: paragraph [0054]), as instantly claimed. Thus, it is considered that Cooper and Tsien collectively disclose each and every additional limitation of amended instant claim 14.
With regard to claim 15, which recites “the athlete [of the method of claim 14] is selected from a human athlete, an equine athlete, a canine athlete and a camelid athlete,” as set forth above, Cooper discloses that in vitro bioassays to detect steroids such as androgens are of increasing importance in combating androgen doping associated with nutritional supplements taken illicitly by athletes to enhance sporting performance and/or physical appearance (Abstract; Section 1. Introduction). Cooper specifically discloses that tetrahydrogestrinone (THG) is a designer androgen (as confirmed by yeast-based in vitro androgen bioassays) found in nutritional supplements that facilitates enhanced sport performance in athletes and can be detected in human urine samples using a bioassay in combination with LC/TOFMS (Section 6.3. Limitation of Structure-Based Methods for Screening of Androgens). Thus, it is considered that Cooper motivates testing a human athlete via the methods collectively disclosed by Cooper, Krusiński, Tsien, and Smith (as evidenced by Slater and Ecehverria) and set forth above, as in instant claim 15.
With regard to amended claim 19, which recites “fluorescence of the reporter construct is measured using static quenching or dynamic quenching,” as set forth above, Krusiński discloses measurement of fluorescence changes in the system taught therein with FRET (Figure 1). Per the instant specification, FRET is a dynamic quenching mechanism (page 24, lines 13-15). Thus, the methods of Krusiński read on the methods of instant claim 19.
With regard to amended claim 20, which recites “fluorescence of the reporter construct is measured using Förster Resonance Energy Transfer,” as set forth above, Krusiński discloses measurement of fluorescence changes in the system taught therein with FRET (Figure 1). Per the instant specification, FRET is a dynamic quenching mechanism (page 24, lines 13-15). Thus, the methods of Krusiński read on the methods of instant claim 20.
With regard to claim 23, which recites “the relative amount of HSP90 to steroid hormone receptor [of the assay method according to claim 13] is x:1, where x is the amount of HSP90 and is defined as [1.0 < x < 5.0],” as set forth above, Smith teaches that cofactors such as HSP90 are required to facilitate and maintain hormone-receptor binding (Page 2229-Paragraph 1; Section "Hormone Binding Ability and Chaperones"-Paragraph 1). In support of this, Echeverria depicts Hsp90 machinery associated with nuclear localization, which includes the species recited in instant claim 5 (i.e. Hsp90, Hsp70, p23, HOP, and Hsp40) in addition to other cofactors (Figure 1). Furthermore, as set forth above, Kang teaches that the ratio of Hsp90 to glucocorticosteroid receptor impacts both receptor-response element binding as well as ligand responsiveness (abstract)
While Kang does not teach the range of ratios disclosed in instant claim 6, they do teach that these ratios impact receptor-ligand interactions, which in turn impact the accuracy of ligand detection using the instantly claimed test kit. Per MPEP 2144.05.II.A, “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)). Thus, given the teachings of Kang regarding the importance of HSP90 to glucocorticosteroid receptor ratio, it would have been obvious to someone of ordinary skill in the art before the filing date of the claimed invention to establish optimum or workable ranges by routine experimentation.
With regard to claim 24, which recites “the relative amount of steroid hormone receptor to nucleic acid reporter construct [of the assay method according to claim 13] is y:1, where y is the amount of steroid hormone receptor and is defined as [1.0 < y < 5.0],” Krusiński discloses experimentation with both nucleic acid reporter construct concentration and protein concentration (i.e. ecdysteroid receptor proteins ultraspiracle (Usp) and ecdysone receptor (EcR)) (Figures 2 and 4; Page 5, Column 2, Paragraph 2-Page 8, Column 1, Paragraph 1). Thus, based on the disclosure of Krusiński, experimentation with the concentration of assay components for optimization thereof constitutes routine experimentation. Per MPEP 2144.05.I.A, “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)). Thus, it is considered that Krusiński discloses each and every additional limitation of instant claim 24.
Given that Cooper, Krusiński, Tsien, and Smith (as evidenced by Slater and Ecehverria) collectively disclose the assay method of instant claim 13, as set forth above, and that Cooper, Krusiński, Tsien, Smith, and Kang disclose the additional limitations of instant claims 14, 19, 20, 23, and 24, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to optimize the ratio of nucleic acid reporter construct to steroid hormone receptor (as disclosed in Krusiński) as well as the ratio of HSP90 to steroid hormone receptor (as motivated by Kang) for purposes of determining the doping status of an athlete, such that analytes (such as steroids) are predictably detected in the tested sample (as motivated by Cooper) by detecting increased or decreased FRET readout (as disclosed in Krusiński and Tsien). One would have been motivated to make such a modification in order to receive the expected benefit of producing an inexpensive and effective assay method for detecting ligands capable of eliciting a steroid hormone genomic response, for example in samples obtained athletes to determine their doping status.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Cooper., 2013 (hereinafter Cooper; as cited in the IDS filed 03/22/2022) in view of Krusiński et al., 2010 (hereinafter Krusiński; of record), US 2006/0112440 A1 (hereinafter Tsien), Smith and Toft, 2008 (hereinafter Smith; of record), and Kang et al., 1999 (hereinafter Kang; of record), as evidenced by Slater and Beato, 1993 (hereinafter Slater; of record) and Echeverria and Picard, 2009 (hereinafter Echeverria; of record), as applied to claims 13 and 14 above, and further in view of the Equine Anti-Doping and Controlled Medication Regulations of the International Federation for Equestrian Sports, 2018 (hereinafter Equine; of record).
The combined disclosures of Cooper, Krusiński, Tsien, Smith, and Kang (as evidenced by Slater and Echeverria) are described above and applied as before. However, these disclosures do not teach the detection of doping in equine athletes, as in instant claim 15.
With regard to claim 15, which recites “the athlete [of the method of claim 14] is selected from a human athlete, an equine athlete, a canine athlete and a camelid athlete,” as set forth above, Cooper, Krusiński, Tsien, Smith, Slater, and Echeverria collectively disclose the assay methods of claims 13 and 14. As previously set forth, Cooper discloses that androgen doping is a concern in human athletes (Abstract; Section 1. Introduction; Section 6.3. Limitation of Structure-Based Methods for Screening of Androgens). However, this concern is not limited to human athletes. Equine discloses that equine athletes are also held to rigorous anti-doping rules (Articles 2.1-2.16) to “preserve what is intrinsically valuable about sport” (Fundamental Rationale for the FEI’s EADCM Regulations (Page 2, Paragraph 1)), thereby motivating testing equine athletes with the methods set forth above. Thus, it is considered that Equine motivates testing an equine athlete via the methods collectively disclosed by Cooper, Krusiński, Tsien, and Smith (as evidenced by Slater and Ecehverria) and set forth above, as in instant claim 15.
As set forth above regarding instant claim 14, given that Cooper, Krusiński, Tsien, and Smith (as evidenced by Slater and Ecehverria) collectively disclose the methods of claims 13 and 14, and that Cooper discloses that androgen doping is a concern in human athletes while Equine discloses that quine athletes are also held to rigorous anti-doping rules, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to apply the assay collectively disclosed by the cited art and set forth above to detect illicit athlete doping (both human and equine) as disclosed in Cooper and Equine to predictably enforce anti-doping policies and ensure fairness in athletic competitions. One would have been motivated to test human and equine athletes using this assay method in order to receive the expected benefit of increased doping detection accuracy.
Claims 17 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Cooper., 2013 (hereinafter Cooper; as cited in the IDS filed 03/22/2022) in view of Krusiński et al., 2010 (hereinafter Krusiński; of record), US 2006/0112440 A1 (hereinafter Tsien), Smith and Toft, 2008 (hereinafter Smith; of record), and Kang et al., 1999 (hereinafter Kang; of record), as evidenced by Slater and Beato, 1993 (hereinafter Slater; of record) and Echeverria and Picard, 2009 (hereinafter Echeverria; of record) as applied to claim 13 above, and further in view of Liu and Lilley, 2017 (hereinafter Liu; of record).
The combined disclosures of Cooper, Krusiński, Tsien, Smith, and Kang (as evidenced by Slater and Echeverria) are described above and applied as before. However, these disclosures do not teach the Cy3 fluorescence generating moiety of instant claim 17 or the Cy5 fluorescence quenching moiety of instant claim 18.
With regard to amended claim 17, which recites “the fluorescence generating moiety [of the test kit according to claim 3] comprises Cy3,” none of the previously cited art discloses Cy3 as a fluorescence generating moiety. This deficiency is cured by Liu, which discloses that Cy3 is extensively used as a fluorescence donor in FRET that has favorable spectroscopic properties and is reasonably photostable (abstract; page 2236, column 1, paragraph 1-page 2236, column 2, paragraph 1). The instantly claimed fluorescence generating moiety is considered to be synonymous with the fluorescence donor disclosed in Liu, as reflected in the instant specification (page 44, lines 10-19). Thus, it is considered that Liu discloses each and every additional limitation of amended instant claim 17.
With regard to amended instant claim 18, which recites “the fluorescence quenching moiety [of the test kit according to claim 3] comprises Cy5, none of the previously cited art discloses Cy5 as a fluorescence quenching moiety. This deficiency is cured by Liu, which discloses that Cy5 is extensively used as a fluorescence acceptor in FRET that has favorable spectroscopic properties and is reasonably photostable (abstract; page 2236, column 1, paragraph 1-page 2236, column 2, paragraph 1). The instantly claimed fluorescence quenching moiety is considered to be synonymous with the fluorescence acceptor disclosed in Liu, as reflected in the instant specification (page 44, lines 10-19). Thus, it is considered that Liu discloses each and every additional limitation of amended instant claim 18.
Given that Cooper, Krusiński, Tsien, and Smith (as evidenced by Slater and Ecehverria) collectively disclose the assay method of instant claim 13, as set forth above, and that Liu discloses that Cy3 and Cy5 are a commonly-used fluorescence donor-acceptor pair in FRET, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize Cy3 and Cy5 (both of which are reasonably photostable and have favorable spectroscopic properties; as disclosed in Liu) as the FRET fluorescence donor-acceptor/generating moiety-quenching moiety pair in the assay method set forth above to predictably and inexpensively detect steroid hormones via their interactions with their associated response elements. One would have been motivated to make such a modification in order to receive the expected benefit of producing an inexpensive and effective assay for detecting ligands capable of eliciting a steroid hormone genomic response with increased detection accuracy using a photostable fluorescence donor-acceptor such as Cy3 and Cy5.
Conclusion
No claims are allowed.
Claims 12 and 13 are objected to.
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/SARAH E ALLEN/ Examiner, Art Unit 1637
/J. E. ANGELL/ Primary Examiner, Art Unit 1637