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 was filed in this application after a decision by the Patent Trial and Appeal Board, but before the filing of a Notice of Appeal to the Court of Appeals for the Federal Circuit or the commencement of a civil action. 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examination under 37 CFR 1.114 and the fee set forth in 37 CFR 1.17(e) has been timely paid, the appeal has been withdrawn pursuant to 37 CFR 1.114 and prosecution in this application has been reopened pursuant to 37 CFR 1.114. Applicant’s submission filed on 05/08/2026 has been entered.
Priority
The present application was filed 11/23/2020, acknowledgement is made of applicant’s claim for foreign priority under 35 U.S.C. 119(a)-(d) to application No 2020-109829, filed 06/25/2020 in Japan.
Information Disclosure Statement
Applicant is reminded that a list of all patents, publications, or other information submitted for consideration by the office must be submitted in a separate paper. The publications of Talucci and Sehlin are not listed in an information disclosure statement.
Appropriate correction is required.
Status of the Claims
Claims 13-14, 16-18, and 20-32 are pending. Claims 13, 18, and 20-22 are amended, claims 1-12, 15, and 19 are canceled and claim 32 is new. Claims 13-14, 16-18, and 20-32 are examined below.
Withdrawn Claim Objections
The objections to claims 13 and 18 are withdrawn due to the amendment of the claims.
The objection to claim 15 is withdrawn due to the cancellation of the claim.
Claim Rejections - 35 USC § 112
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.
Claims 16 and 17 are 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.
Claims 16 and 17 are indefinite, at the recitation of claim 15, because claim 15 is cancelled. The claims are further indefinite because there is insufficient antecedent basis for “the labeling substance” in claim 16 and “the enzyme” in claim 17. No prior recitation of a labelling substance or an enzyme is recited.
In the interest of compact prosecution claim 16 is examined as being dependent on claim 18 and claim 17 is being examined as being dependent on claim 16.
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 13-14, 16-18 and 20-31 are rejected under 35 U.S.C. 103 as being unpatentable over Lejbman et al. (10/14/2016) “Active duty service members who sustain a traumatic brain injury have chronically elevated peripheral concentrations of Aβ40 and lower ratios of Aβ42/40” Brain injury, 30, 12, pages 1436-1441 (see PTO-892, 07/17/2026), in view of Rissin et al. (06/2010) “Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations”, Nature biotechnology, 28, 6, pages: 595-599 (see PTO-892, 07/17/2026), Kleinschmidt et al., US 2011/0091910A1 (see PTO-892, 03/02/2023) and Yamaguchi et al., US 2008/0025988A1, 01/31/2008 (see PTO-892, 07/17/2026).
Regarding claim 18, Lejbman teaches a method for measuring Aβ40 and Aβ42 concentrations in plasma samples using an ultrasensitive single-molecule enzyme-linked immunosorbent assay (SIMOA), which is a paramagnetic bead-based ELISA, comprising monoclonal anti-Aβ40 and -Aβ42 capture antibody directed to the N-terminus (6E10) and biotinylated detector antibodies ADx Neurosciences ADx specific for Aβ40 and clone H31L21 specific for Aβ42, both directed to the C-terminus (Lejbman, abstract and page 1437, ‘Measures’, line 1-page 1438, line 3). Lejbman further teaches the concentration of Aβ40 and Aβ42 in pg/mL, for example the amount of Aβ40 in the traumatic brain injury (TBI) negative group is about 150 pg/mL. As such Lejbman teaches an amount (150 pg) per milliliter of blood (Lejbman, page 1439, see figure 1 (a)). Lejbman indicate that this assay is based on SIMOA technology (page 1437, left column).
Lejbman does not include full details of their SIMOA assay method.
However, Rissin teaches further details of the SIMOA assay format, used for detecting serum proteins at subfemtomolar concentrations (Rissin, see Abstract), comprising beads functionalized with an antibody to the target protein and incubating samples with the beads. Rissin further teaches separating and washing the beads, after which they are incubated with solutions containing detection antibody. After another wash step the beads, are loaded into a well array (Rissin, page 599 +1, ‘Online Methods’, see ‘Capture of proteins on magnetic beads and formation of enzyme-labeled immunocomplex’). The sample comprising the enzyme-labeled beads is placed in contact with a substrate and fluorescence is measured (Rissin, page 599 +1, ‘Online Methods’, ‘Detection of beads and enzyme-labeled beads in femtoliter-volume well arrays’, lines 5-9). In short, the SIMOA assay comprises mixing the blood sample, which according to the specification on page 7, paragraph [0009] can be whole blood, plasma, or serum, with a capture antibody, a solid phase (beads), and a detection antibody forming a solid phase complex, which is then analyzed (Lejbman, page 1436, 2nd paragraph, lines 16-17). Rissin teaches that the ability to detect single protein molecules in blood could accelerate discovery and use of more sensitive diagnostic biomarkers and that the SIMOA assay uses fluorescence imaging to detect single protein molecules. Rissin further teaches that this digital ELISA approach detects as few as 10-20 enzyme-labeled complexes in a sample and allows detection of clinically relevant proteins in serum at concentrations much lower than conventional ELISA (Rissin, page 595, see Abstract).
It would have been prima facie obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to have incorporated the known procedural details of the SIMOA method, as taught by Rissin, when performing the SIMOA assay of Lejbman, in particular by incubating samples with antibody functionalized beads, and after separating and washing, further incubating the samples with detection antibody, followed by washing and placing the beads into a well array, where samples are contacted with substrate and fluorescence is measured. The ordinary artisan would have been motivated to do so, because the SIMOA allows for detection of single protein molecules in blood which accelerates discovery and use of more sensitive diagnostic biomarkers, because clinically relevant proteins can be detected at much lower concentrations than in a conventional assay such as an ELISA. That is, Lejbman teaches a SIMOA assay method but simply does not provide the full procedural details of this technique, however, it would have been obvious to consult Rissin who do provide such further details.
Lejbman also differs from the claimed invention as it does not teach a capture antibody that is clone 82E1. Instead, Lejbman exemplifies 6E10 as the capture antibody, describing 6E10 as an antibody directed to the N-terminus of Aβ42 and Aβ40.
Kleinschmidt teaches the 8E21 antibody, which is described as an antibody recognizing the N-terminus of Aβ40/42, (Kleinschmidt, page 8, see paragraphs [0096], [0157-160], and claim 13). Kleinschmidt further teaches that the 8E21 antibody can be used as an immobilized antibody (i.e., a capture antibody) in a method of detecting full length Aβ42 in blood, serum, or plasma by ELISA [0029], [0157]-[0160].
Yamaguchi also teaches the 8E21 antibody, indicating that this monoclonal antibody recognizes the N-terminus peptide of amyloid β while not recognizing amyloid β precursor proteins (Yamaguchi, Abstract, line 1-3). In particular, Yamaguchi specifically teaches that antibody clone 82E1 does not react with amyloid β (2-40) and amyloid β (3-40), but only amyloid β (1-40) and also does not react with amyloid β precursor proteins. Yamaguchi further teaches that in contrast to 82E1, clone 6E10 but recognizes amyloid β (2-40) and (3-40) and also recognizes amyloid β precursor proteins (APP; Yamaguchi, page 7, see paragraphs [0093]-[0095]; [0005]; Figs. 2-3).
Thus, Yamaguchi teaches that the 82E1 antibody is more specific to the N-terminus of amyloid β peptide as compared with the 6E10 antibody.
It would have been prima facie obvious to one having ordinary skill before the effective filing date of the claimed invention, to have modified the method of Lejbman and Rissin by substituting the antibody clone 6E10 with the antibody clone 82E1, because Kleinschmidt indicate that 82E1, like 6E10 of Lejbman, was also known in the prior art to be an N-terminal antibody specific to the common N-terminus of Aβ40/42 and also suitable for use as a capture antibody in an ELISA assay for Aβ42, which is the same purpose for which 6E10 was used by Lejbman. One would have been further motivated to substitute 8E21 for 6E10 because Yamaguchi taught that unlike clone 6E10, clone 82E1 does not recognize amyloid β precursor proteins.
Accordingly, one of ordinary skill in the art would have been motivated to substitute 8E21 for 6E10 in order to perform the same purpose of serving as a capture antibody directed to the N-terminus of Aβ40 and Aβ42 in the method of Lejbman, while providing increased specificity for the N-terminus since unlike 6E10, 8E21 would not cross-react with related, non-target molecules such as the APP precursor. Using such an antibody that is more specific for the N-terminus of Aβ40/Aβ42 would have been desirable since the purpose of Lejbman’s assay is to specifically detect Aβ40 and Aβ42, not the precursor.
The ordinary artisan would have a reasonable expectation of success, because both Lejbman and Kleinschmidt teach a sandwich assay comprising a capture and detection antibody and enzyme linked detection of Aβ42 in blood samples and Kleinschmidt teaches success using the antibody clone 82E1 for detection of amyloid β.
Regarding claim 13, Lejbman indicate that their SIMOA assay uses a capture antibody on paramagnetic beads (i.e., solid phase). Rissin similarly teaches that in the SIMOA assay, beads were functionalized with antibody to the target protein (Rissin, page 599 +1, ‘Online Methods’, ‘Capture of proteins on magnetic beads and formation of enzyme-labeled immunocomplex’, lines 2-3).
Regarding claim 14¸ Lejbman teaches that the method is a paramagnetic bead assay (Lejbman, page 1437, ‘Measures’, lines 2-3).
Regarding claims 16 and 17¸ Lejbman teaches a biotinylated detector antibody (Lejbman, page 1437-1438, ‘Measures’, lines 5-1), but does not provide full details of their assay and is silent on what ultimate detection label was used, but as above do identify it as SIMOA technology. Rissin teaches a SIMOA assay using the enzyme conjugate streptavidin-β-galactosidase which binds the detection antibody with the biotin tag (Rissin, page 596, see figure 1a, ‘2.’ and ‘3.’). In summary, Lejbman teaches a SIMOA assay comprising a biotinylated antibody and Rissin indicate that in the SIMOA assay the biotinylated antibody is then bound by an enzyme. Accordingly, when performing the SIMOA assay of Lejbman, it would have been obvious to consult Rissin for further procedural details of this assay and to have used the biotinylated antibody in conjunction with an enzyme label (claim 16), specifically a β-galactosidase enzyme label (claim 17) in order to successfully carry out the SIMOA assay according to the known techniques of Rissin.
Regarding claim 31, Lejbman teaches determining Aβ42 in plasma samples by single-molecule enzyme-linked immunosorbent assay (SIMOA). A SIMOA assay (as further detailed by Rissin) comprises beads functionalized with an antibody to the target protein and incubating samples with the beads. It further teaches separating and washing the beads, incubating the bead with solutions containing detection antibody and after another wash step, loading the beads into a well array (not an immune complex transfer method; Rissin, page 599 +1, ‘Online Methods’, see ‘Capture of proteins on magnetic beads and formation of enzyme-labeled immunocomplex’). The sample comprising the enzyme-labeled beads are placed in contact with a substrate and fluorescence is measured (Rissin, page 599 +1, ‘Online Methods’, ‘Detection of beads and enzyme-labeled beads in femtoliter-volume well arrays’, lines 5-9). In short, the SIMOA assay method of Lejbman and Rissin does not comprise transferring the complex from the solid phase to another solid phase.
Regarding claim 20, Lejbman teaches a method for measuring Aβ40 and Aβ42 concentrations in plasma samples using and ultrasensitive single-molecule enzyme-linked immunosorbent assay (SIMOA), comprising monoclonal anti-Aβ40 and -Aβ42 capture antibody directed to the N-terminus (6E10) and biotinylated detector antibodies ADx Neurosciences ADx specific for Aβ40 and clone H31L21 specific for Aβ42, both directed to the C-terminus (Lejbman, page 1437, ‘Measures’, line 1-page 1438, line 3).
As discussed previously in detail above, the combined teachings of Lejbman, Rissin, Kleinschmidt, and Yamaguchi fairly suggest a method for measuring Aβ42 in blood using a capture antibody that is clone 82E1 and a detection antibody which is an H31L21 antibody. Briefly, Lejbman teaches a method for measuring Aβ40 and Aβ42 concentrations in plasma samples using a paramagnetic bead-based ELISA, comprising monoclonal anti-Aβ40 and -Aβ42 capture antibody directed to the N-terminus (6E10) and biotinylated detector antibodies ADx Neurosciences ADx specific for Aβ40 and clone H31L21 specific for Aβ42, both directed to the C-terminus (Lejbman, page 1437, ‘Measures’, line 1-page 1438, line 3). Lejbman further teaches the concentration of Aβ40 and Aβ42 in pg/mL, for example the amount of Aβ40 in the traumatic brain injury (TBI) negative group is about 150 pg/mL. As such Lejbman teaches an amount (150 pg) per milliliter of blood (Lejbman, page 1439, see figure 1 (a)).
Lejbman does not provide full details of their assay and is silent on what ultimate detection label was used. Rissin teaches that the SIMOA assay comprises beads functionalized with an antibody to the target protein and further teaches incubating samples with the beads. It further teaches separating and washing the beads, after which they are incubated with solutions containing detection antibody. After another wash step the beads are subsequently loaded into a well array (Rissin, page 599 +1, ‘Online Methods’, see ‘Capture of proteins on magnetic beads and formation of enzyme-labeled immunocomplex’). The sample comprising the enzyme-labeled beads are placed in contact with a substrate and fluorescence is measured (Rissin, page 599 +1, ‘Online Methods’, ‘Detection of beads and enzyme-labeled beads in femtoliter-volume well arrays’, lines 5-9).
As such it meets the limitation “wherein the method is not an immune complex transfer method” when interpreted in light of the specification and the prior art (see Claim Interpretation, above). Lejbman in view of Rissin on the other hand teaches a method where beads functionalized with an antibody are incubated with samples, followed by washing the beads and incubating them with detection antibody. After a further wash step, the antibody coated beads comprising the analyte and detection antibody are loaded into a well array (Rissin, page 599 +1, ‘Online Methods’, see ‘Capture of proteins on magnetic beads and formation of enzyme-labeled immunocomplex’) and contacted with an enzyme substrate and fluorescence is measured (Rissin, page 599 +1, ‘Online Methods’, ‘Detection of beads and enzyme-labeled beads in femtoliter-volume well arrays’, lines 5-9), i.e. without transferring the immune complex to another substrate.
Lejbman does not include full details of their assay method. However, as discussed previously in detail above, the SIMOA assay of Lejbman in view of Rissin teaches mixing the blood sample with a capture antibody, a solid phase (beads), and a detection antibody forming a solid phase complex, which is then analyzed (Lejbman, page 1436, 2nd paragraph, lines 16-17).
Lejbman also fails to specifically teach a capture antibody that is clone 82E1 (for detection of Aβ42), nor a detection antibody that is 1A10.
Kleinschmidt teaches a sandwich ELISA which comprises a pair of capture and detection antibodies which are specific for a region of Aβ(1-40) (detection antibody; Kleinschmidt, page 8, paragraph [0153], lines 6-11). Kleinschmidt teaches a detection antibody, 1A10 (Kleinschmidt, page 8, paragraph [0159], line 3), which is a C-terminal specific for an epitope in the region of amino acid residues 35-40 of Aβ(1-40) (Kleinschmidt, [0159]; page 23, claim 13, line 38). As discussed in further detail with respect to claim 18 above, Kleinschmidt also teaches the 82E1 antibody recognizing the N-terminus of Aβ40 and Aβ42 (Kleinschmidt, page 8, see especially paragraphs [0157-160])
It would have been prima facie obvious to one having ordinary skill before the effective filing date of the claimed invention, to have modified the invention of Lejbman in order to use the antibody clone 1A10 as taught by Kleinschmidt in place of the detection antibody ADx Neurosciences ADx of Lejbman as an obvious matter of a simple substitution of one art recognized detection antibody for another, both recognized as suitable for the same purpose, both binding the C-terminal end of Aβ40 in blood. The ordinarily skilled artisan would have been motivated to do so, because the 1A10 antibody of Kleinschmidt, performs the same function in substantially the same way and produces substantially the same results of detecting the C-terminal end of Aβ40 in blood. In substituting one antibody recognizing the Aβ40 C-terminus (ADx Neurosciences ADx of Lejbman) with that of Kleinschmidt (1A10) known to have the same binding specificity, one would have had an expectation of success because both are taught in the art to be used for immunoassays detecting Aβ40 in blood (same target, same sample). As such the modification would yield a predictable result.
With respect to the 82E1 capture antibody, as discussed in detail above with respect to claim 18, Kleinschmidt teach this antibody and Yamaguchi specifically teaches that 82E1 does not react with amyloid β (2-40) and amyloid β (3-40), but only amyloid β (1-40) and also does not react with amyloid β precursor proteins.
As discussed in detail above with respect to claim 18, it would have been prima facie obvious to one having ordinary skill before the effective filing date of the claimed invention, to have modified the method of Lejbman by substituting the antibody clone 6E10 with the antibody clone 82E1, because of the teaching of Kleinschmidt of using clone 82E1 as a capture antibody in an ELISA assay for Aβ42 and the teaching of Yamaguchi that unlike clone 6E10, clone 82E1 does not recognize amyloid β precursor proteins. Thus in addition to selecting 82E1 as another known N-terminal antibody known to be suitable for the same purpose of 6E10, the ordinary artisan also would have been motivated to choose 82E1 as it would have been expected to be more specific for the intended target molecule.
The ordinary artisan would have a reasonable expectation of success, because both Lejbman and Kleinschmidt teach a sandwich assay comprising a capture and detection antibody and enzyme linked detection of Aβ42 in blood samples and Kleinschmidt teaches success using the antibody clone 82E1 for detection of amyloid β.
Regarding claims 21 and 22, Lejbman and the cited art above teach a method substantially as claimed. Both Lejbman and Rissin teach immobilizing the capture antibody on a solid phase (see, e.g., Lejbman, page 1437, ‘Measures’, lines 2-3 teaching paramagnetic beads).
Lejbman in view of Rissin does not teach a capture antibody that is clone 82E1.
As discussed previously in detail above, Kleinschmidt teaches a method of detecting full length Aβ40 and Aβ42 by ELISA with an immobilized antibody recognizing the N-terminus of Aβ42, comprising clone 82E1 (Kleinschmidt, page 8, see paragraphs [0157-158]). Kleinschmidt further teaches detecting Aβ42 in blood, serum or plasma (Kleinschmidt, page 3, paragraph [0029]).
Yamaguchi teaches a monoclonal antibody recognizing the N-terminus peptide of amyloid β while not recognizing amyloid β precursor proteins (Yamaguchi, Abstract, line 1-3). Yamaguchi specifically teaches that the antibody clone 82E1 only reacts with amyloid β (1-40) and also does not react with amyloid β precursor proteins. Yamaguchi further teaches that in contrast to 82E1, clone 6E10 also recognizes amyloid β precursor proteins (Yamaguchi, page 7, see paragraph [0095]).
It would have been prima facie obvious to one having ordinary skill before the effective filing date of the claimed invention, to have modified the method of Lejbman in view of Rissin by replacing the antibody clone 6E10 with the antibody clone 82E1, because of the teaching of Kleinschmidt of using clone 82E1 as a detection antibody in an ELISA assay for Aβ42 and the teaching of Yamaguchi that unlike clone 6E10, clone 82E1 does not recognize amyloid β precursor proteins.
The ordinary artisan would have a reasonable expectation of success, because both Lejbman and Kleinschmidt teach a sandwich assay comprising a capture and detection antibody and enzyme linked detection of Aβ42 in blood samples and Kleinschmidt teaches success using the antibody clone 82E1 for detection of amyloid β.
Regarding claim 23 and 24¸ Lejbman teaches that the method is a paramagnetic bead assay (Lejbman, page 1437, ‘Measures’, lines 2-3).
Regarding claims 25 and 26¸ Lejbman teaches that the detection antibodies are biotinylated (Lejbman, page 1437-1438, ‘Measures’, lines 5-1).
Regarding claims 27-30¸ Lejbman teaches a biotinylated detector antibody (see above), but does not provide full details of their assay and is silent on what ultimate detection label was used, but as above do identify it as SIMOA technology. Rissin teaches a SIMOA assay using the enzyme conjugate streptavidin-β-galactosidase which binds the detection antibody with the biotin tag (Rissin, page 596, see figure 1a, ‘2.’ and ‘3.’). In summary, Lejbman teaches a SIMOA assay comprising a biotinylated antibody and Rissin indicate that in the SIMOA assay the biotinylated antibody is then bound by an enzyme. Accordingly, when performing the SIMOA assay of Lejbman, it would have been obvious to consult Rissin for further procedural details of this assay and to use the biotinylated antibody in conjunction with an enzyme label (claim 16), specifically a β-galactosidase enzyme label (claim 17) in order to successfully carry out the SIMOA assay according to the known techniques of Rissin.
Claim 32 is rejected under 35 U.S.C. 103 as being unpatentable over Lejbman et al., in view of Rissin et al., Kleinschmidt et al., Yamaguchi et al., Andreasson et al. (2018) “Commutability of the certified reference materials for the standardization of β-amyloid 1-42 assay in human cerebrospinal fluid: lessons for tau and β-amyloid 1-40 measurements”, Clinical Chemistry and Laboratory Medicine, 56, 12, pages 2058-2066 (see PTO-892, 03/02/2023), Portelius et al. Characterization of amyloid β peptides in cerebrospinal fluid by an automated immunoprecipitation procedure followed by mass spectrometry. Journal of proteome research. 2007 Nov 2;6(11):4433-9, Lu et al., 1997, “Effects of acetonitrile on horseradish peroxidase (HRP)-anti HRP antibody interaction”, Biosensors & Bioelectronics, 12, 7, pages 619-625 (see PTO-892, 07/17/2026), Chambers et al., 2017, “Development of a Quantitative SPE/LC/MS/MS Assay for Multiple Amyloid Beta Peptides in Cerebrospinal Fluid”, https://www.waters.com/webassets/cms/library/docs/720003600en.pdf (Poster; see PTO-892, 07/17/2026)), and Gu et al., US9856315B2.
Regarding claim 32, Lejbman in view of Rissin, Kleinschmidt, and Yamaguchi teach a method of measuring an amount of Aβ using and immunoassay comprising a capture antibody 82E1 and a detection antibody H31L21 substantially as claimed (see above, the rejection of claim 18).
Lejbman and the cited art above fails to teach that the values measured by immunoassay (value X) and those measured by mass spectrometry (value Y) exhibit a correlation coefficient r of 0.8 or more. Lejbman further fails to teach a mass spectrometry method comprising immunoprecipitation of the Aβ peptide using anti-Aβ antibody 6E10, releasing the peptide and antibody with a solution containing 0.56% ammonia and 40% acetonitrile. Lejbman further fails to teach separating the solution by liquid chromatography and measuring the Aβ peptide using electrospray ionization using multiple reaction monitoring in positive ion measurement mode and that the MRM transitions are precursor ion/product ion is precursor ion/product ion of 1129.5/1078.8.
Andreasson teaches using different methods analyzing Aβ42 and Aβ40 in cerebrospinal fluid samples and certified reference materials including different immunoassays and mass spectrometry (Andreasson, page 2061, ‘Assays’, lines 1-8). Andreasson further teaches that there are four biomarkers for Alzheimer’s disease, including Aβ42 and Aβ40 which are included in research criteria but ideally uniform global cut-off levels will be established to facilitate a more general use, for example in routine clinical diagnostics. However, this is hindered by the lack of harmonization between different method and laboratories (Andreasson, page 2058, ‘Introduction’, line 4-page 2059, line 11). Andreasson further teaches that non-linear association between different methods might pose a problem for the harmonization of global diagnostic cutoffs and manufacturers should take measures to ensure that their methods have linear associations with the reference measurement procedures (Andreasson, page 2064, lines 3-7). Andreasson further teaches comparing all immunoassays to liquid chromatography-tandem mass spectrometry (see figures 1-3) and as such teaches mass spectrometry as the reference method.
Andreasson does not teach separating the target from the antibody nor the specifics of mass spectrometry.
Portelius teaches an optimized Aβ immunoprecipitation-mass spectrometry method that enables accurate and rapid monitoring of the major Aβ isoforms (Portelius, Abstract, lines 4-5), automating the washing and elution procedure which improves the speed of sample preparation and, due to crosslinking of antibodies, eliminates the problem of interfering antibodies that otherwise are coeluted with the antigen (Portelius, page 4433, 2nd column, 4th paragraph, line 1-page 4434, line 2). Portelius teaches immunoprecipitating the cerebrospinal fluid sample with Aβ specific 6E10 antibody (Portelius, page 4434, ‘Sample preparation’, lines 1-4) and then transferring the beads/CSF solution to an automated washing and elution of peptides comprising eluting the Aβ peptides using 0.5% formic acid (Portelius, page 4434, see entire 4th paragraph). Portelius further teaches analyzing the samples by mass spectrometry (Portelius, see title).
Portelius does not teach 0.56% ammonia and 40% acetonitrile for elution.
Lu et al. teach a 40% acetonitrile buffer and the effect of acetonitrile concentration on antibody-antigen binding. Lu further teaches that in solutions containing between 40% and 80% acetonitrile, an irreversible decrease in binding was observed, most likely due to irreversible changes in the conformation of the antibody (Lu et al., page 619, Abstract, lines 10-15).
Chambers et al. teaches an acetonitrile extraction buffer comprising NH4OH (Chambers et al, Experimental, Solid Phase Extraction (SPE), Elute, 75:15:10 ACN:H2O: NH04) for solid phase elution, though it fails to teach 0.56 %. Chambers also teaches that solid phase elution was one of the more critical aspects of the overall methodology (Chambers et al., Solid Phase Extraction, paragraph 3) and that steps were taken at all stages of the methodology to avoid Aβ specific challenges such as non-specific binding, poor solubility, and aggregation. Chambers et al. further teaches liquid chromatography using an ACQUITY UPLC BEH C18 system (Chambers et al., Experimental, see UPLC Method Conditions) and then measuring the Aβ peptide using Xevo TQ triple quadrupole mass spectrometry operated in electrospray ionization (Chambers et al., Experimental, MS Conditions) and positive ion mode (Chambers et al., Results and Discussion, see Mass Spectrometry). Chambers et al. further teaches that the mass spectrometer MRM transitions are precursor ion/product ion 1083/1054 and 1129/1079 for Aβ40 and Aβ42 peptide respectively (Chambers et al., Table 1). Chambers et al. also teach that the analysis of Aβ peptides is extremely challenging, not only because of the relatively low abundance in biological fluids, but also because of their propensity for aggregation with other proteins and themselves. LC/MS/MS with the proper sample preparation described here is capable of recovering pg/ml levels of amyloid peptides in the presence of high concentrations of interfering proteins and peptides. The method is also high throughput, reliable and capable of quantitating multiple peptides in a class.
Gu et al. is cited as another example in the art recognizing ammonia/acetonitrile solution for its art recognized ability to elute bound peptides, see specifically column 42, lines 30-36, Gu similarly discloses this combination (ammonia and acetonitrile) recognized for elution, Gu teaching 2.5% ammonia/50% acetonitrile as a basic elution solution (different concentration ratios than Chambers, thereby supporting concentration of the reagents as optimizable variables; Gu, column 42, lines 30-36).
It would have been prima facie obvious to one having ordinary skill at the time the claimed invention was effectively filed to have compared the method of Lejbman and the prior art above with a reference mass spectrometry method as taught by Andreasson because of the teaching of Andreasson that ensuring that different methods have linear associations with the reference measurement procedures helps with harmonization between different methods and establishing uniform global cut-off levels which facilitate a more general use, for example in routine clinical diagnostics.
It would have further been prima facie obvious to one having ordinary skill at the time the claimed invention was effectively filed to have modified the method of the reference mass spectrometry as taught by Andreasson with the immunoprecipitation mass spectrometry as taught by Portelius because of the teaching of Portelius that this enables accurate and rapid monitoring of the major Aβ isoforms and eliminates the problem of interfering antibodies that otherwise are coeluted with the antigen.
It would have been further prima facie obvious to one having ordinary skill at the time the claimed invention was effectively filed to have used a buffer comprising acetonitrile and ammonia as the elution buffer (elution buffer following immunoprecipitation in order to release AB), in order to prevent rebinding of the antibody and maintain the pH, particularly because these reagents were known in the art at the time for their ability to elute bound complex (Lu and Chambers) . The ordinary artisan would have been motivated to rely on solution containing acetonitrile and ammonia in order to retrieve the maximum possible amount of peptide from the sample, particularly considering the art supports these reagents as recognized for, and capable of, eluting Aβ peptides.
One of ordinary skill in the art would have a reasonable expectation of success comparing the results of the immunoassay as claimed with mass spectrometry because of the teaching of Andreasson of comparing immunoassays to reference mass spectrometry.
One of ordinary skill in the art would have a reasonable expectation of success applying the immunoprecipitation and antigen elution method of Portelius to the method of Andreasson because Portelius teaches success using the method in a mass spectrometry assay and Andreasson teaches mass spectrometry.
Further, one having ordinary skill would have a reasonable expectation of success applying the specific mass spectrometry method as taught by Chambers as the reference method of Andreasson because Andreasson teaches mass spectrometry for the detection of Aβ, though Andreasson is silent on the specifics of the method, and Chambers teaches a mass spectrometry method that is optimized for the detection of Aβ peptides.
Regarding the claimed percentages of acetonitrile and ammonia, it would have been a further obvious matter of routine optimization, to have arrived at the buffer comprising 40% acetonitrile and .56% ammonia for the following reasons.
See MPEP 2144.05, Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
In the present case, the concentrations of the reagents, ammonia and acetonitrile, are result effective variables, namely these variables achieve the art recognized results of elution. See as cited previously above, 40% acetonitrile was recognized previously in the art as appropriate for elution, Lu specifically teaching that between a range of 40% and 80% acetonitrile, an irreversible decrease in binding was observed. It would have been obvious, and well within the skill level of the ordinary artisan to have arrived at the claimed 40% out of routine optimization of experimental conditions, namely varying the concentration between the art disclosed range, to uncover the optimum workable concentration in the solution for elution.
This is similarly the case for the concentration of ammonia, although the prior art fails to specifically disclose .56% ammonia, Chambers teach a concentration that is 10% (see as discussed above, 75:15:10 ACN:H2O: NH04), and Gu teach 2.5% ammonia and 50% acetonitrile. Considering the different prior art teach different concentrations (close in magnitude) and usable for release/elution, it would have been well within the skill level of the ordinary artisan to have optimized the concentration of ammonia to uncover the optimum workable percentage to achieve elution, particularly in combination with acetonitrile, given that both acetonitrile and ammonia together in solution was recognized at the time as usable for this purpose (for the purpose of elution).
Further, one having ordinary skill would have a reasonable expectation of success optimizing the concentrations, particularly considering the prior art recognized different concentrations for elution (for example, Chambers using a different concentration combination from Gu).
Further, it would have been obvious to perform liquid chromatography using an ACQUITY UPLC BEH C18 system as claimed and then measuring the Aβ peptide using Xevo TQ triple quadrupole mass spectrometry operated in electrospray ionization in positive ion mode, with the mass spectrometer MRM transitions values for precursor ion/product ion being 1083/1054 and 1129/1079 for Aβ40 and Aβ42 peptide respectively (as claimed), because Chambers et al. teach that this method is specifically optimized at every step to minimize or eliminate the impact of Aβ specific issue, such as low abundance in biological fluids and their propensity for aggregation with other proteins and themselves.
The ordinary artisan would have been motivated to use a buffer as outlined above and then LC/MS/MS with the proper sample preparation described here, because it was known capable at the time of recovering pg/ml levels of amyloid peptides in the presence of high concentrations of interfering proteins and peptides. The method is also high throughput, reliable and capable of quantitating multiple peptides in a class.
Regarding the correlation coefficient r of 0.8 or more between a measured value X and a measured value Y obtained by the method and a reference mass spectrometry method, the limitation does not add a manipulative difference, rather it states the desired result without adding a method step and therefore does not result in a patentable difference over the prior art, the prior art teaching all active methods steps recited.
Response to Arguments
Applicant's arguments filed 05/08/2026 have been fully considered but they are not persuasive.
Applicant argues, starting on page 8 that the results demonstrated by applicant are unexpected. Applicant argues that data shows poor correlation between the results of immunoassays and mass spectrometry for the detection of Aβ42 in blood. Applicant further argues that the results of an immunoassay using the same pair of antibodies as taught by Lejbman (6E10 and H31L21) did not correlate well with mass spectrometry unlike the antibody pair of the present application which shows unexpectedly high correlation. Applicant remarks that the examiner and the board assert that these results are not unexpected because Yamaguchi discloses that the 82E1 antibody is more specific for the N-terminus of Aβ40/Aβ42.
This argument is not persuasive.
Yamaguchi does not teach that the 82E1 antibody is more specific for the N-terminus but rather that this monoclonal antibody recognizes the N-terminus peptide of amyloid β while not recognizing amyloid β precursor proteins (Yamaguchi, Abstract, line 1-3). Further, Yamaguchi further teaches that in contrast to 82E1, clone 6E10 but recognizes amyloid β (2-40) and (3-40) and also recognizes amyloid β precursor proteins (emphasis by examiner; Yamaguchi, page 7, see paragraphs [0093]-[0095]; [0005]; Figs. 2-3).
As such, Yamaguchi’s teachings refer to the proteins bound by the antibody via the N-terminus, but not, as recited by the applicant, that the antibody is “more specific for the N-terminus”.
Applicant further argues that Talucci (Talucci et al. Aggregation-dependent epitope sequence and modification fingerprints of anti-Aβ antibodies. Elife. 2026 Feb 9;14:RP106156) demonstrated that 82E1 antibody detected Aβ(2-x) and several N-terminally elongated Aβ peptides in addition to Aβ(1-x). Applicant argues that 82E1 antibody is a commercial product and it is therefore understandable that Yamaguchi asserts high specificity of this antibody but that a fair and independent evaluation by a third party demonstrates a disadvantage of the 82E1 antibody relative to the 6E10 antibody disclosed in Kleinschmidt.
This argument is not persuasive.
Talucci examines specific binding epitopes for anti-Aβ antibodies using peptide libraries (Talucci, page 3 of 32, ‘Pre-screening of Aβ-Ab epitopes’, lines 1-4). Talucci uses a library of 15-mer peptides overlapping in 14 amino acids and probes of Aβ-antibody binding (Talucci, page 5 of 32, see Figure 1 legend). Talucci further recites that the initial microarray analysis defined linear binding motifs but needs to be complemented in different setting, as more complex features of the binding behavior may be missed otherwise and then recites that in the following sections the focus is shifted to full length Aβ peptides which should reflect the conformations encountered in vivo and therefore provide insights into relevant antibody binding (Talucci, page 13 of 32, lines 1-5). As such, Talucci points out that the epitope screening microarray (said screening array using 15-mer peptides rather than the native proteins) needs to be complemented by assays using the full length protein in order to test the conformation encountered in vivo. Talucci does not screen binding of 82E1 to full length protein. The observation by Talucci that 82E1 binds multiple short peptide residues does not contradict the observation of Yamaguchi that 82E1, when tested against full-length proteins (rather than short peptide epitopes as in Talucci) recognizes the N-terminus peptide of amyloid β while not recognizing amyloid β precursor proteins unlike clone 6E10 which recognizes amyloid β (2-40) and (3-40) and also recognizes amyloid β precursor proteins (see above).
Applicant further argues (page 10) that Sehlin reaches that 82E1 produces non-specific signals produced by heterophilic antibodies in an ELISA assay making 82E1 less desirable.
This argument is not persuasive.
Sehlin (Sehlin, D., 2010. Aβ Conformation Dependent Antibodies and Alzheimer's Disease (Doctoral dissertation, Acta Universitatis Upsaliensis) recites that in an ELISA using 82E1 as both capture and detection antibody showed interference by heterophilic antibodies (Sehlin, page 32, 2nd paragraph, lines 1-4). Sehlin further teaches that samples are likely to be susceptible to heterophilic antibody interference, especially if the capture and detection antibodies are from the same species (Sehlin, page 32, 8-10). Sehlin does not recite any other antibodies and as such does not teach that 82E1 is especially susceptible to the interference by heterophilic antibodies compared to other Aβ-antibodies. Further Sehlin recites multiple strategies which can be used to mitigate the effects of heterophilic antibodies such as dilution of the sample and the use of a commercially available buffer designed to neutralize heterophilic antibodies eliminating the signal (Sehlin, page 32, 2nd paragraph, lines 7-9). Applicant does not recite compared to what antibody 82E1 is less desirable and the teaching of Sehlin does not compare 82E1 to other Aβ-antibodies but rather uses it as an example in an assay where both capture and detection antibodies are 83E1 and as such are of the same species. Put another way, the interference by heterophilic antibodies is a problem in assays using capture and detection antibodies derived from the same species and not an 83E1 specific problem and therefore does not make 83E1 less desirable than other antibodies.
Applicant further argues that the unexpected superior results of the claimed antibody expectation (as demonstrated by the correlation with mass spectrometry results) should be given weight even if mass spectrometry is not a limitation of the claims but rather is used to show the superiority of the claimed antibody pair.
This argument is not persuasive.
As explained previously in detail above, the results are not unexpected due to the different protein binding profiles of the antibodies as taught in the prior art.
Applicant further argues, starting on page 11, that new claim 32 recites a correlation coefficient r of 0.8 or more between the method and a reference mass spectrometry method.
As explained previously in detail above, the value of the correlation coefficient, i.e. 0.8 or more, is not an active method step and therefore does not result in a patentable difference over the prior art.
For all the reasons above, the arguments are not persuasive.
Conclusion
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/STEFANIE J. KIRWIN/Examiner, Art Unit 1677
/Soren Harward/Primary Examiner, TC 1600
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