DETAILED ACTION
Applicant’s amendment filed 4/7/2025 is acknowledged. Claim 1 is currently amended. Claims 3-5, 9-10, 20-26, and 28-30 are canceled. Claims 14-19 and 27 remain withdrawn pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention. Claims 1-2, 6-8, 11-19, and 27 remain pending in the instant application.
Claims 1-2, 6-8, and 11-13 are the subject of this Office 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 8/6/2025 has been entered.
Priority
The instant application claims priority to U.S. Provisional Application No. 63/030,481 filed on May 27th, 2020.
Response to Amendment
Applicant’s amendment to Claim 1 overcomes the objection and 35 U.S.C. § 112(b) rejection previously set forth in the Final Rejection mailed on 2/6/2025. Accordingly, the rejection is withdrawn.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 1-2, 6-8, and 11 are rejected under 35 U.S.C. 103 as being unpatentable over Fleischman et al. (US2019/0064165; of record) in view of Zhang et al. (US2016/0040209; of record), Pocker et al. (J. Am. Chem. Soc. 1982, Vol. 104, p.4885-4889; of record), Yabuki et al. (Sensors and Actuators 2000, Vol. 65, p.49-51; of record), Bekhit et al. (Anal. Chem. 2019, Vol. 91, p.3163-3169; of record in the IDS filed 1/19/2022), and Schwaderer et al. (WO2016/161413; of record).
Regarding claims 1-2 and 11, Fleischman teaches an electrochemical method for detecting leukocyte esterase (LE) activity in a sample using the internally calibrated electrochemical continuous enzyme assay (ICECEA) (see paragraphs [0002], [0006], [0022], [0039], and [0076]-[0079]). Fleischman teaches that a LE substrate (see Fleischman claim 1) that can be hydrolyzed by LE to generate a redox intermediate can be used to generate a detectable chemical response (see paragraphs [0042]-[0043]). Fleischman generally describes that the ICECEA process includes electrodes (see paragraphs [0022] and [0077]). Additionally, Fleischman teaches that leukocyte esterase is an enzyme present in urine samples and is used as an indicator of an infectious process (see paragraph [0003]).
Fleischman does not teach contacting the sample with an anti-LE antibody coated surface forming an immobilized LE from the sample; contacting the immobilized LE with an assay solution comprising methyl pyruvate (LE substrate) and alcohol oxidase to form a test solution; and electrochemically measuring concentration of H2O2 produced in the test solution using a nitrogen doped carbon nanotube (N-CNT) electrode at a negative detection potential.
Zhang teaches a method for determining enzyme activity using the ICECEA method (see Abstract and paragraphs [0008]-[0011] and [0036]-[0053]). Zhang teaches that the ICECEA method can use different redox species to monitor the progress of enzymatic reaction by following their generation and specifically suggests H2O2 (see paragraph [0053]). Zhang teaches measuring H2O2 concentration when measuring esterase enzyme activity (see paragraphs [0098]-[0099] and Figures 15-16). The hydrolysis of the esterase substrate yields an alcohol that is then oxidized by an oxidase redox enzyme (see paragraph [0106]). Zhang teaches the redox enzyme is immobilized in a thin polymeric film on the surface of an electrode (see paragraphs [0090] and [0098]).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention to have combined the method for determining leukocyte activity in a sample using the ICECEA method as taught by Fleischman with the method for detecting enzyme activity of an esterase by measuring H2O2 concentration using the ICECEA method with an oxidase redox enzyme immobilized on an electrode as taught by Zhang. One of ordinary skill in the art would have been motivated to because Zhang provides specific details on the measurement of esterase activity by measuring the H2O2 concentration produced in a redox reaction using a redox enzyme and it would have yielded predictable results. One of ordinary skill in the art would have had a reasonable expectation of success because Fleischman suggests that redox intermediates can be used to detect the activity of LE.
Zhang does not teach contacting the sample with an anti-LE antibody coated surface forming an immobilized LE from the sample, contacting the immobilized LE with an assay solution comprising methyl pyruvate (LE substrate) and alcohol oxidase, or using a nitrogen doped carbon nanotube (N-CNT) electrode at a negative detection potential.
Pocker teaches that esterases can be probed using the hydrolysis of methyl pyruvate (see Abstract and p.4885, left column, first paragraph). The hydrolysis of methyl pyruvate provides methanol, an alcohol, and pyruvate.
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have substituted methyl pyruvate, as taught by Pocker, for the LE substrate used in the ICECEA method of detecting LE activity, as taught by Fleischman as modified by Zhang above. One of ordinary skill in the art would have been motivated to because the hydrolysis of methyl pyruvate by esterase is known to create an alcohol intermediate useful in the redox reaction for the detection of LE activity of Fleischman as modified by Zhang, yielding predictable results. One of ordinary skill in the art would have had a reasonable expectation of success because Fleischman as modified by Zhang utilize the hydrolysis of the LE substrate into an alcohol to measure the activity of the enzyme from subsequent redox reactions.
Pocker does not teach contacting the sample with an anti-LE antibody coated surface forming an immobilized LE from the sample, an alcohol oxidase, or using a nitrogen doped carbon nanotube (N-CNT) electrode at a negative detection potential.
Yabuki teaches a peroxidase enzyme immobilized to a glassy carbon electrode but suggests that combining it with an alcohol oxidase can yield highly-sensitive biosensors (see p.50, right column, last passage, and p.51, left column, first passage). Yabuki also teaches that H2O2 is measured by redox reactions (see p.49, left and right columns).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have substituted a glassy carbon electrode with alcohol oxidase combined with peroxidase immobilized to its surface as disclosed by Yabuki for the immobilized enzyme electrode of Fleischman as modified by Zhang above. One of ordinary skill in the art would have been motivated to use a glassy carbon electrode with alcohol oxidase combined with peroxidase immobilized on its surface to have a highly-sensitive biosensor for H2O2 in the method for detecting LE activity in a sample of Fleischman as modified by Zhang. One of ordinary skill would have been motivated to use a highly-sensitive biosensor for H2O2 because Fleischman as modified by Zhang teach detecting H2O2 concentration to detect LE activity. One of ordinary skill in the art would have had a reasonable expectation of success because each of Zhang and Yabuki teach measuring H2O2 produced by redox reactions using a redox enzyme immobilized on the surface of an electrode.
Yabuki does not teach contacting the sample with an anti-LE antibody coated surface forming an immobilized LE from the sample or using a nitrogen doped carbon nanotube (N-CNT) electrode at a low negative detection potential.
Bekhit teaches an ICECEA-dipstick immunoassay for myeloperoxidase detection which comprises immobilizing the enzyme myeloperoxidase in saliva samples on a dipstick comprising an antibody specific for myeloperoxidase and using the dipstick with the immobilized myeloperoxidase in the ICECEA method (see Abstract, p.3164, right column, first-last paragraphs, and p.3167, left column, last paragraph, and right column). Bekhit further teaches immobilizing an antibody specific for myeloperoxidase to a dipstick. Bekhit teaches that the capture of myeloperoxidase on the dipstick resulted in a percentage of recovery of 101%, to effectively separate the enzyme of interest, myeloperoxidase, from saliva samples. Myeloperoxidase is an enzyme of interest found in saliva as it is an indicator of a wide range of inflammatory and autoimmune diseases. One of ordinary skill in the art recognizes that the ICECEA-dipstick immunoassay for myeloperoxidase taught by Bekhit can be modified for the detection of other enzymes of interest simply by substituting an antibody specific for the enzyme of interest for the antibody immobilized on the dipstick. Bekhit additionally teaches the use of a nitrogen-doped carbon nanotube (N-CNT) electrode for the measurement of H2O2 at -0.20 V, which could detect 0.50 μM H2O2, reads on at a negative detection potential, and provides the procedure for nitrogen doping a glassy carbon electrode (see Abstract and p.3164, left column, 5th-last paragraph bridging left and right columns). Thus, the electrode detects low amounts of H2O2 and is sensitive for H2O2.
Bekhit does not teach an anti-LE antibody.
Schwaderer teaches an antibody that is specific for leukocyte esterase (see paragraph [0006], [0017], and [0064]). This reads on an anti-LE antibody as claimed. Schwaderer further teaches the antibody-marker complex can be measured by electrochemical methods including voltammetry and amperometry methods (see paragraph [0054]).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have substituted the antibody specific for leukocyte esterase as taught by Schwaderer for the antibody immobilized on the dipstick of Bekhit and to use this dipstick in an ICECEA assay for leukocyte esterase as taught by Fleischman as modified by Zhang, Pocker, and Yabuki above and to nitrogen dope the electrode used and use it at -0.20 V, to arrive at the claimed invention. One of ordinary skill in the art would be motivated to because leukocyte esterase is an enzyme of interest and indicator of infectious process and Schwaderer teaches the antibodies are useful in electrochemical methods, yielding predictable results. One of ordinary skill in the art would be motivated to immobilize an antibody specific for leukocyte esterase on the dipstick for ICECEA immunoassay since it was shown to effectively separate the enzyme of interest from a test sample for use in ICECEA. One of ordinary skill in the art would have been motivated to nitrogen dope the electrode used and to use it at a -0.20 V detection potential because Bekhit teaches its use in an ICECEA assay, which Fleischman and Zhang are directed to as well, and the detection of low amounts of H2O2. Thus, the electrode is sensitive for H2O2. One of ordinary skill in the art would have had a reasonable expectation of success since Fleischman, Zhang, and Bekhit are directed to ICECEA enzyme assays.
Thus, a method for detecting leukocyte esterase (LE) activity in a sample, the method comprising: (a) contacting the sample with an anti-LE antibody coated surface forming an immobilized LE from the sample; (b) contacting the immobilized LE with an assay solution comprising methyl pyruvate and alcohol oxidase to form a test solution; and (c) electrochemically measuring concentration of H2O2 produced in the test solution using a nitrogen doped carbon nanotube (N-CNT) electrode at a -0.20 V detection potential (i.e. a negative detection potential), as recited in claim 1, is prima facie obvious over Fleischman in view of Zhang, Pocker, Yabuki, Bekhit, and Schwaderer. The teachings above also read on claims 2 and 11.
Regarding claims 6 and 8, Fleischman discloses that the ICECEAs generally follow the method described in U.S. Patent Publication No. 2016/0040209 (see paragraphs [0075]-[0076]). Fleischman teaches three distinct calibration steps performed in the pre-assay phase by spiking a solution of enzyme substrate and necessary reactants with a solution of the redox active component of the enzymatic reaction and measuring an increase in current flowing through the electrode in contact with the enzyme substrate and necessary reactants spiked with a redox active component (see paragraphs [0080] and [0086] and Fig. 4). The electrode is calibrated prior to contacting the test sample with the electrode. One of ordinary skill in the art recognizes that a solution of enzyme substrate and necessary reactants would include the redox enzyme, which as discussed above in the rejection of claim 1, is immobilized on the surface of the electrode. Thus, Fleischman teaches the electrode is calibrated prior to contacting the test sample with the electrode, comprising contacting the electrode with a calibration sample, spiking the calibration sample with aliquots of the redox active component of the enzymatic reaction, and measuring an increase in current flowing through the electrode in contact with the calibration sample. The calibration sample being the assay solution (enzyme substrate and redox enzyme) prior to contacting with the sample. One of ordinary skill in the art recognizes that methyl pyruvate is the enzyme substrate, alcohol oxidase is the redox enzyme, and H2O2 is the redox active component of the enzymatic reaction, as discussed in the rejection of claim 1 above.
Fleischman does not disclose spiking the calibration sample with aliquots of H2O2.
Zhang teaches spiking a calibration sample with aliquots of 2.0 µM H2O2 during calibration in ICECEA when determining the activity of an esterase (see paragraphs [0098]-[0099] and Fig. 15).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have used 2.0 µM H2O2 aliquots for spiking the calibration sample as taught by Zhang to calibrate the electrode for the redox active component of the enzymatic reaction of Fleischman. One of ordinary skill in the art would have been motivated because Zhang teaches using H2O2 aliquots for spiking when measuring the activity of esterases, and H2O2 is the redox active component of the redox reaction for esterases. One of ordinary skill in the art would have had a reasonable expectation of success because leukocyte esterase is an esterase.
Regarding claim 7, Fleischman, Zhang, Pocker, Yabuki and Schwaderer do not disclose the calibration sample can be spiked with 50-µL aliquots of 0.200 mM H2O2 solution, per 5 mL of calibration solution.
Bekhit teaches spiking a calibration sample with 50-µL aliquots of H2O2 in a 5 mL solution in the ICECEA method (p.3164, left column, last passage, right column, first passage). Thus, Bekhit teaches spiking a calibration sample with 50-µL aliquots of H2O2 per 5 mL of calibration solution.
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have combined spiking a calibration sample with aliquots of 2.0 µM H2O2 as taught by Zhang above in the rejection of claim 6 with spiking a calibration solution with 50-µL aliquots of H2O2 per 5 mL of calibration solution as taught by Bekhit. One of ordinary skill in the art would have been combining the known elements of spiking calibration samples with H2O2 as taught by Zhang with using 50-µL aliquots in a 5 mL calibration solution to yield predictable results. One of ordinary skill in the art would have had a reasonable expectation of success because each of Fleischman, Zhang, and Bekhit are directed to ICECEA enzyme assays.
Neither Fleischman nor Bekhit teach a 0.200 mM H2O2 concentration aliquot for spiking during calibration.
Zhang teaches a 0.200 mM H2O2 concentration aliquot for spiking during calibration (see paragraphs [0080]-[0081]).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have used 0.200 mM H2O2 concentration aliquots for spiking during calibration as taught by Zhang to spike the calibration sample of Fleischman. One of ordinary skill in the art would have been motivated to use 0.200 mM H2O2 concentration aliquots because they would have been applying known calibration methods in the ICECEA assay. One of ordinary skill in the art would have had a reasonable expectation of success because Zhang used the 0.200 mM H2O2 aliquots to calibrate the ICECEA assay for H2O2 detection which Fleischman and Bekhit also detect through the ICECEA assay.
Thus, claims 6-8 are prima facie obvious over Fleischman in view of Zhang, Pocker, Yabuki, Bekhit, and Schwaderer.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Fleischman et al. (US2019/0064165; of record) in view of Zhang et al. (US2016/0040209; of record), Pocker et al. (J. Am. Chem. Soc. 1982, Vol. 104, p.4885-4889; of record), Yabuki et al. (Sensors and Actuators 2000, Vol. 65, p.49-51; of record), Bekhit et al. (Anal. Chem. 2019, Vol. 91, p.3163-3169; of record in IDS filed 1/19/2022), and Schwaderer et al. (WO2016/161413; of record), as applied to claims 1-2, 6-8, and 11 above, and further in view of Gorski et al. (WO2019/094575; of record).
Fleischman in view of Zhang, Pocker, Yabuki, Bekhit and Schwaderer teach the invention of claim 1 as discussed in the rejection above.
Regarding claim 12, Fleischman, Zhang, Pocker, Yabuki, Bekhit and Schwaderer do not disclose that the assay solution comprises 10 mg/L to 2000 mg/L of methyl pyruvate.
Gorski teaches the concentration of leukocyte esterase substrate in the test sample can be between 10 mg/L to 2000 mg/L (see paragraph [0014]). One of ordinary skill in the art recognizes that the concentration of methyl pyruvate in the test sample would not differ from the concentration of methyl pyruvate in the assay solution, absent evidence to the contrary. Thus, Gorski teaches the concentration of leukocyte esterase substrate in the test sample can be between 10 mg/L to 2000 mg/L.
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have combined Gorski’s teaching for using a concentration of substrate between 10 mg/L to 2000 mg/L with Fleischman’s method, as modified by Zhang, Pocker, Yabuki, Bekhit and Schwaderer above, for detecting leukocyte esterase activity using methyl pyruvate as the substrate, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated because Gorski teaches a concentration range for leukocyte esterase substrates and methyl pyruvate is an esterase substrate. One of ordinary skill in the art would have had a reasonable expectation of success because each of Fleischman, Zhang, Bekhit, and Gorski are directed to the ICECEA assay. Therefore, claim 12 is prima facie obvious over Fleischman in view of Zhang, Pocker, Yabuki, Bekhit, and Schwaderer and further in view of Gorski.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Fleischman et al. (US2019/0064165; of record) in view of Zhang et al. (US2016/0040209; of record), Pocker et al. (J. Am. Chem. Soc. 1982, Vol. 104, p.4885-4889; of record), Yabuki et al. (Sensors and Actuators 2000, Vol. 65, p.49-51; of record), Bekhit et al. (Anal. Chem. 2019, Vol. 91, p.3163-3169; of record in IDS filed 1/19/2022), and Schwaderer et al. (WO2016/161413; of record), as applied to claims 1-2, 6-8, and 11 above, and further in view of Bourdillon et al. (J. Am. Chem. Soc. 1980, Vol. 102, p.4231–4235; of record).
Fleischman in view of Zhang, Pocker, Yabuki, Bekhit and Schwaderer teach the invention of claim 1 as discussed in the rejection above.
Regarding claim 13, Fleischman, Zhang, Pocker, Yabuki, Bekhit and Schwaderer do not disclose that the assay solution comprises 10 mg/L to 2000 mg/L of alcohol oxidase.
Bourdillon teaches immobilizing 1 mg/mL (1000 mg/L) glucose oxidase on a glassy carbon electrode for measuring H2O2 (see p.4232, left column, fourth paragraph). 1000 mg/L falls within the claimed range of 10 mg/L to 2000 mg/L.
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have immobilized alcohol oxidase on a glassy carbon electrode doped with nitrogen, as taught by Fleischman as modified by Zhang, Pocker, Yabuki, and Bekhit above, at a concentration of 1000 mg/L as taught by Bourdillon. One of ordinary skill in the art would have had a reasonable expectation of success because they would have been using the known enzyme concentration of 1000 mg/L for immobilization and it would have yielded predictable results in H2O2 measurement. Therefore, claim 13 is prima facie obvious over Fleischman in view of Zhang, Pocker, Yabuki, Bekhit, and Schwaderer and further in view of Bourdillon.
Response to Arguments
Applicant's arguments filed 4/7/2025 have been fully considered but they are not persuasive.
In Applicant’s Remarks, see p.1, last paragraph-p.2, last paragraph, Applicant submits that that Bekhit establishes that antibodies can interfere with the enzymatic activity of their target enzymes. Applicant argues that the current claim provides for an enzymatically active LE/antibody complex and that Schwaderer teaches a detection antibody and not an anti-LE antibody that retains enzymatic activity as claimed. Applicant argues that Schwaderer discloses a colorimetric assay to detect the presence of a protein or peptide, not an assay of LE’s enzymatic activity, and thus the Action fails to establish that the Schwaderer LE/antibody complex retains enzymatic activity. This is not found persuasive. Applicant has not provided evidence that the LE-specific antibodies disclosed in Schwaderer would not allow for LE to retain enzymatic activity. See MPEP § 2145(I) which sets forth that argument does not replace evidence where evidence is needed. The instant specification describes that any known LE antibody of the prior art can be an anti-LE antibody (see paragraphs [0011]-[0012]). Schwaderer teaches an antibody specific for LE and thus reads on an anti-LE antibody of the claimed invention. Furthermore, Schwaderer teaches the antibody-marker complexes can be measured by electrochemical methods (see paragraph [0054]). Thus, it is expected that the anti-LE antibody of Schwaderer would remain enzymatically active in an electrochemical method of Fleischman in view of Zhang, Pocker, Yabuki, and Bekhit.
In Applicant’s Remarks, see p.3, 2nd and last paragraphs, Applicant argues that claims 12 and 13 are not obvious since claim 1 is nonobvious. This is not found persuasive for the reasons discussed in the reply above and the rejections are maintained.
Conclusion
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/J.P.S./Examiner, Art Unit 1657
/MELENIE L GORDON/Supervisory Patent Examiner, Art Unit 1657