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 .
Status of the Claims
Claims 1-8 are pending and examined herein.
Priority
The present application, filed 07/25/2024, is a 371 of PCT/CZ2022/050009, filed 01/31/2022, which claims foreign priority of CZPV 2022-34, filed 01/25/2022. Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Rejections - 35 USC § 112
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 8 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 enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The enablement requirement is such that the specification of the described invention in such terms that one skilled in the art can make and use the invention to ensure that the invention is communicated to the interested public in a meaningful way. (MPEP 2164)
The standard for determining whether the specification meets the enablement requirement is determined in view of the Wands factors (MPEP 2164.01(a)), to assess whether any necessary experimentation required by the specification is "reasonable" or is "undue." The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation needed to make or use the invention based on the content of the disclosure, (2) the amount of direction provided by the inventor, (3) the existence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the level of one of ordinary skill, (7) the level of predictability in the art, and (8) the breadth of the claims. While all of these factors are considered a sufficient amount for a prima facie case are discussed below.
The breadth of the claim
Claim 8 depends from claim 1 and requires not merely quantitative detection of MUC1fs, but the use of the immunoassay measurements to determine structural characteristics of the MUC1fs VNTR. Specifically, claim 8 requires “determination of concentration of MUC1fs in the sample, determination of the VNTR length, and determination of the mutation position,” wherein “a length of mutated region of VNTR MUC1fs in sample is determined using an appropriate calibration curve obtained by analyzing a standard containing at least one epitope of the mutated form of VNTR MUC1fs” and “a length of wild-type region of VNTR MUC1fs in the sample is determined using an appropriate calibration curve obtained by analyzing a standard containing at least one epitope of the wild-type form of VNTR MUC1fs.” Thus, the claimed function is not accomplished merely by detecting the presence of MUC1fs or measuring antibody-binding signal. Claim 8 requires the measured responses obtained using standards containing mutant and wild-type VNTR epitopes to permit determination of the length of the mutated region, the length of the wild-type region, the overall VNTR length, and the mutation position in MUC1fs present in the biological sample.
The breadth of this determination is significant in view of Applicant’s own description of MUC1. The specification states that the number of VNTR repeats is highly variable, typically ranging from 20 to 125 repetitions, and each repeat is approximately 60 ± 3n base pairs long, and further states that the repeats are subject to polymorphism ([0002], p. 1). The specification further explains that the mutation may occur within the VNTR such that MUC1fs generally contains a wild-type portion and a mutated portion, with the relative lengths of those portions depending upon the position of the mutation ([0003], p. 1).
Indeed, Applicant acknowledges the underlying structural variability by stating that due to the high variability caused by the different length of the VNTR region and the possibility of the mutation itself being anywhere within this region, it is impossible to detect ADTKD-MUC1 in a wide range of variants by standard genetic testing methods. and therefore it is very difficult to diagnose [0005], p. 2). Claim 8 nevertheless encompasses determining these structural characteristics from the recited calibration-curve immunoassay without limiting the claim to a particular total VNTR length, particular number of wild-type or mutated repeats, particular mutation position, particular MUC1fs molecular concentration, or experimentally validated relationship between antibody response and VNTR-region length.
The nature of the invention
The invention concerns an immunochemical method that uses quantitative antibody measurements as a basis for determining structural characteristics of a highly repetitive MUC1fs molecule. Applicant describes a system comprising an immobilized antibody against mutated VNTR MUC1fs together with antibodies directed against the wild-type and mutated VNTR portions. The specification states that by comparing both normalized obtained signals, it is possible to determine, among other things, the position of the mutation and the relative length of individual forms of VNTR ([0007], pp. 3-4). The method requires a skilled artisan to use the quantitative antibody responses to infer structural information concerning the MUC1fs molecule—namely, how much of the VNTR lies before and after the frameshift and, from those determinations, the VNTR length and mutation position. Accordingly, the artisan must know how those measurements correspond quantitatively to the lengths of the respective regions of the MUC1fs molecule.
The amount of direction provided by the inventor / The existence of working examples
Applicant provides substantial direction concerning how to perform the immunoassay and obtain quantitative antibody measurements, but does not provide corresponding experimental direction demonstrating how those measurements are converted into the structural determinations required by claim 8. The specification describes dividing the sample into two portions and detecting the respective VNTR forms. For the wild-type VNTR portion, Applicant identifies an antibody recognizing APDTRP (SEQ ID NO. 2), followed by an antibody against anti-wt-VNTR (anti-anti-wt-VNTR) carrying horseradish peroxidase (HRP) and spectrophotometric detection; for the mutated VNTR portion, Applicant identifies an antibody recognizing SPRCHLGPGHQAGPGLHRRP (SEQ ID NO. 1), likewise followed by antibody against anti-fs-VNTR (anti-anti-fs-VNTR) carrying HRP and spectrophotometric detection ([0008], p. 4).
Applicant also expressly provides a calibration approach. The specification states that the calibration curve is obtained using a specially developed standard based on bovine serum albumine (BSA) carrying antigenic epitopes of MUC1 and MUC1fs, including GVTSAPDTRPAPG for the wild-type VNTR and SPRCHLGPGHQAGPGLHRRP for the mutated VNTR ([0009], pp. 4-5). Applicant then states that concentration of anti-wt-VNTR and anti-fs-VNTR antibodies determined by quantification using the calibration curve corresponds to concentrations of non-mutated and mutated MUC1fs repeats in the examined biological sample ([0009], p. 4-5). Applicant further states that, following normalization and quantification, it is not only possible to determine the concentration of MUC1fs in a biological sample in any variant, i.e. regardless of VNTR length or mutation position, but it is also possible to determine the VNTR length and mutation position in the variant present in a specific sample ([0010], p. 5).
However, the specification does not provide the quantitative relationship necessary to perform the latter structural determinations. It does not provide an equation, mathematical model, lookup table, experimentally established signal-to-repeat-number relationship, or other quantitative procedure showing how a measured concentration or absorbance corresponding to the wild-type epitope is converted into the number or length of wild-type VNTR repeats on a MUC1fs molecule, or how the corresponding mutated-VNTR measurement is converted into the number or length of mutated repeats. The qualitative guidance of paragraph [0010] does not resolve this deficiency. Applicant describes low and high combinations of anti-wild-type and anti-mutated VNTR measurements as corresponding generally to a mutation near the C-terminus, near the N-terminus, or in the middle of the VNTR region ([0010]-[0011], p. 5). Such qualitative categories do not teach the artisan how to determine the length of either VNTR region or how the quantitative measurements are converted into the mutation-position determination required by claim 8.
The drawings likewise illustrate the proposed concept but do not experimentally establish the missing quantitative relationship. Figure 1 schematically labels separate measurements for determination of length of fs-VNTR and determination of length of wt-VNTR, with the mutation represented between the two regions (Fig. 1; [0015], p. 6). Figure 2 schematically illustrates the modified BSA calibration standard (Fig. 2; [0016], p. 6), while Figure 3 depicts the conceptual relationship between anti-fs-VNTR and anti-wt-VNTR measurements and different MUC1fs configurations (Fig. 3; [0017], p. 6). These drawings provide a schematic model of the asserted relationship; they do not supply experimental data showing that particular measured values correspond to particular known VNTR lengths or known mutation positions.
Example 1 similarly demonstrates the assay procedure rather than the structural inference required by claim 8. Applicant describes immobilizing an anti-MUC1fs antibody recognizing SEQ ID NO. 1, analyzing plasma, detecting the wild-type and mutant portions with the respective antibodies, developing the signal with TMB and hydrogen peroxide, and measuring absorbance at 450 nm ([0025]-[0026], p. 7-8). Applicant also assigns calibration values, including 16-fold dilution corresponds to 100 U/ml of wild-type VNTR repeats and 256-fold dilution corresponds to 100 U/ml of mutated VNTR repeats ([0026], p. 8).
Those disclosures demonstrate that quantitative immunoassay measurements can be obtained. They do not demonstrate that a particular U/ml measurement establishes a particular number of tandem repeats per MUC1fs molecule, the length of the wild-type or mutated region of an individual MUC1fs molecule, or the position of the frameshift within the VNTR. The remaining examples and Figures 4–10 likewise report quantitative anti-wild-type and/or anti-mutated VNTR measurements in controls, disease populations, or during patient monitoring, but do not identify samples having independently established VNTR lengths and mutation positions and then demonstrate that Applicant’s immunoassay correctly recovers those structural characteristics. Thus, the specification provides a working immunoassay and calibration procedure, but does not provide a working example demonstrating the particular signal-to-structure inference required by claim 8. The deficiency is therefore not an inability to measure MUC1fs epitopes. Rather, the missing teaching is how those measurements reliably determine VNTR-region length and mutation position.
The state of the prior art / The level of predictability in the art
The state of the prior art reinforces, rather than supplies, the quantitative relationship missing from Applicant’s disclosure. Wenzel et al. (Single molecule real time sequencing in ADTKD-MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations, Scientific Reports. Vol. 8, No. 1, March 2018), is particularly informative regarding the structural variability and technical difficulty associated with determining MUC1 VNTR architecture. Wenzel explains that the VNTR size of each parental MUC1 allele is highly polymorphic (usually between 20 to 125 units per allele) and further explains that the VNTR comprises almost identical 60-mer repeat units (p. 2, Fig. 1). Wenzel additionally explains that a previously developed probe-extension assay had the loss of almost all structural and positional information, prompting the authors to employ targeted single-molecule real-time sequencing (SMRT) to generate complete VNTR assemblies (p. 3).
Wenzel’s results further demonstrate substantial positional variability. The authors report that the precise location of the 60-mer unit location harbouring the insC within the MUC1-VNTR could be solved in eight of the nine positive families studied and that position of the insC ranged from the second up to the 39th unit (p. 6). Moreover, even two families having the mutation in the 39th variable unit had different VNTR assembly and allele size (p. 6). Wenzel therefore demonstrates that VNTR length and mutation position are determinable structural characteristics, but determines those characteristics through complete VNTR assembly, rather than by assuming that detection or magnitude of a mutant-repeat signal itself provides the positional information. Indeed, Wenzel expressly identifies loss of almost all structural and positional information as a limitation of an assay capable of detecting the mutant repeat. This evidence is relevant because claim 8 similarly requires more than detection or quantification of mutant and wild-type epitopes: it requires those measurements to yield the underlying VNTR-region lengths and mutation position.
Karsten et al. (Binding patterns of DTR-specific antibodies reveal a glycosylation-conditioned tumor-specific epitope of the epithelial mucin (MUC1). Glycobiology. Vol. 14, No. 8. August 2004) provides additional evidence concerning the predictability of using MUC1 antibody signal as a quantitative proxy for repeat length. Karsten expressly examined sole and combined effects of peptide length (number of repeats) and O-glycosylation with GalNAc at the DTR motif on the binding patterns of 22 monoclonal antibodies recognizing this motif (p. 681). The authors found that in case of unglycosylated peptides, almost all antibodies bound better to multiple MUC1 tandem repeats. Glycosylation at the DTR led to enhanced binding in 11 cases, whereas 10 antibodies were not influenced in binding, and one was inhibited. In nine of the former cases both length and DTR glycosylation were additive in their influence on antibody binding, suggesting that both effects are different. (p. 681).
More significantly, when Karsten directly evaluated length dependency, the authors found that almost all antibodies showed increased binding toward the longer peptides, but this increase differed considerably among individual antibodies and that a subgroup was almost nonreactive with the single repeat but revealed significant and increasing binding with increased numbers of repeats (p. 682). Karsten further determined that the length and glycosylation effects were independently variable. The authors report that the increment in absorbance varies from strong to weak, and there are also cases where no additional length effect can be observed, and that nine of eleven antibodies in one group showed an additive influence of glycosylation and length, clearly indicating that both effects are different (p. 683, Fig. 1). Figure 1 itself shows different antibody-response profiles as the number of MUC1 repeats increases from one to five (p. 684, Fig. 1). Karsten’s discussion summarizes the point particularly clearly: the authors examined antibody binding with respect to peptide length (i.e., the number of tandem repeats) and DTR glycosylation and concluded that the impacts of length and glycosylation on antibody binding, which at first hand appeared to be similar, were in fact different (and in a subgroup of mAbs additive) (p. 689). The authors additionally observed that even nominally similar antibodies reveal astonishing differences in their fine specificity (p. 689). Karsten is particularly relevant because Applicant’s wild-type epitope is APDTRP (SEQ ID NO. 2), whereas Karsten specifically investigated DTR-directed MUC1 antibodies and identifies multiple antibodies having APDTR/APDTRP-containing epitopes.
Thus, the prior art does not establish a universal relationship in which a particular increase in antibody signal necessarily corresponds to a particular increase in VNTR repeat number. Instead, Karsten provides objective evidence that MUC1 antibody response to repeat multiplicity can vary depending upon the particular antibody and glycosylation state. Accordingly, Wenzel demonstrates substantial structural variability in MUC1 VNTR length and mutation position and the need to resolve VNTR architecture to obtain exact positional information, while Karsten demonstrates that MUC1 antibody binding itself is affected in a nonuniform manner by tandem-repeat number and glycosylation. Neither reference supplies the quantitative signal-to-structure relationship omitted from Applicant’s specification. Rather, the state of the art demonstrates that such a relationship cannot simply be presumed from the ability to obtain quantitative antibody signals. These factors weigh strongly against enablement.
The level of ordinary skill in the art
The level of ordinary skill in the relevant art would have been relatively high (PhD and/or MD) and would reasonably have included substantial knowledge of immunoassays, antibody-antigen interactions, MUC1 biology, molecular biology, protein biochemistry, and analysis of tandem-repeat proteins. Such an artisan could readily perform the underlying laboratory operations disclosed by Applicant. Applicant itself provides sufficient practical details for immobilizing an anti-MUC1fs antibody, contacting the captured material with wild-type- and mutant-specific antibodies, performing HRP/TMB detection, measuring absorbance, and generating calibration measurements. The deficiency therefore is not technical inability to perform an immunoassay or generate a calibration curve.
Rather, the missing information is the quantitative structural relationship that claim 8 requires the artisan to assign to the resulting measurements. High skill in immunoassay methodology does not itself tell the artisan whether, for example, a given anti-mutated-VNTR signal represents a greater number of MUC1fs molecules containing relatively few mutant repeats, fewer MUC1fs molecules containing relatively many mutant repeats, differences in antibody-binding behavior associated with repeat multiplicity or glycosylation, or some combination thereof. Karsten’s experimentally observed differences in antibody response with repeat number and glycosylation reinforce that this issue cannot be resolved merely through proficiency in performing the assay.
Likewise, high skill does not supply the missing rule for converting the resulting mutant and wild-type measurements into a particular VNTR length and mutation position. Wenzel demonstrates that exact mutation position can be established when the underlying VNTR architecture is reconstructed, but does not establish that a pair of quantitative epitope measurements inherently provides that architecture. Accordingly, the relatively high level of ordinary skill does not overcome the missing teaching.
The quantity of experimentation needed to make or use the invention based on the content of the disclosure
The experimentation necessary to establish and practice the full scope of the claimed structural determination would be substantial. Applicant has not supplied experimental data from samples having independently established MUC1 VNTR lengths and mutation positions from which the skilled artisan could derive and validate the claimed relationship. Therefore, rather than merely optimizing a known calibration curve, the artisan would first have to establish whether and under what conditions the two immunoassay measurements quantitatively encode the structural characteristics required by claim 8. At a minimum, the artisan would have to obtain or generate a representative panel of MUC1fs molecules or biological samples having independently established total VNTR lengths and independently established mutation positions; determine the corresponding numbers or lengths of wild-type and frameshifted VNTR repeats; perform the claimed anti-wild-type and anti-mutated VNTR immunoassays across those structurally characterized samples; determine how changes in MUC1fs molecular concentration affect the two signals independently of repeat number; determine the response of the particular antibodies as the number of corresponding VNTR epitopes changes; account for relevant differences in antibody recognition, including the length- and glycosylation-dependent effects demonstrated by Karsten; derive a quantitative relationship capable of distinguishing molecular concentration from repeat number; establish how the two measurements are combined to calculate total VNTR length and mutation position; and validate that relationship across VNTR lengths and mutation positions encompassed by the claim.
Wenzel illustrates why this is not merely confirmation of a structural relationship already supplied by the art. The MUC1 VNTR is highly polymorphic (usually between 20 to 125 units per allele), and the mutation position in Wenzel’s positive families extended from the second up to the 39th unit. Karsten independently demonstrates why generating a calibration curve using an epitope-containing standard does not by itself resolve the issue: the increase differed considerably among individual antibodies as repeat number increased, while glycosylation produced antibody-dependent effects on binding. Consequently, the artisan would not merely be optimizing known assay parameters. The artisan would be required to generate and validate the quantitative signal-to-structure relationship that is itself necessary to make the claimed determination operative.
The calibration curves disclosed by Applicant do not remove this experimentation. A calibration curve generated using a standard containing an epitope can establish a relationship between assay response and the amount of that standard under the calibration conditions. It does not, absent further teaching or validation, establish that a measured response from a biological sample uniquely specifies the number of corresponding repeats on each captured MUC1fs molecule. Nor does it establish how the resulting wild-type and mutant measurements are converted into the claimed VNTR length and mutation position. Considering the limited quantitative guidance provided by the specification, the absence of a working example validating VNTR length or mutation position from samples of independently known structure, the substantial VNTR structural variability demonstrated by Wenzel, the nonuniform effects of repeat length and glycosylation upon MUC1 antibody binding demonstrated by Karsten, and the empirical work that would be necessary to establish and validate the missing signal-to-structure relationship across the scope of claim 8, the quantity and nature of the required experimentation are undue.
Considering the Wands factors as a whole, claim 8 is not enabled. Applicant expressly describes the desired result and provides sufficient technical guidance for obtaining quantitative anti-wild-type and anti-mutated MUC1fs immunoassay measurements. However, the specification does not provide sufficient teaching demonstrating how those measurements are converted into the length of the mutated VNTR region, the length of the wild-type VNTR region, the resulting VNTR length, and the mutation position required by claim 8. Applicant’s calibration standard and Example 1 demonstrate quantitative immunoassay measurement, but do not establish a quantitative relationship between a particular measured value and a particular number or length of VNTR repeats on an individual MUC1fs molecule. The drawings schematically depict the asserted structural determination, but do not provide experimental validation using samples of independently known VNTR length and mutation position. The state of the art does not supply the missing teaching. Wenzel demonstrates that MUC1 VNTR architecture is highly polymorphic and that exact positional information was obtained by complete VNTR assembly, while also identifying the loss of almost all structural and positional information associated with an assay directed to detecting the mutant repeat. Karsten further demonstrates that MUC1 antibody response does not exhibit a universally predictable relationship with repeat number, because the increase differed considerably among individual antibodies and antibody binding is additionally affected by glycosylation.
Accordingly, the deficiency is not an inability to perform the disclosed immunoassay or obtain quantitative antibody measurements. Rather, the specification fails to provide sufficient guidance, supported by working evidence or predictable knowledge in the art, that would permit the skilled artisan to translate those measurements into the structural determinations required by claim 8 without first undertaking substantial empirical investigation to establish and validate that relationship. Therefore, given the breadth and nature of the claimed structural determination, the limited guidance concerning the conversion of immunoassay signal to VNTR structure, the absence of a working structural-determination example, the variability of MUC1 VNTR architecture and antibody binding demonstrated by the prior art, and the substantial experimentation necessary to establish the missing quantitative relationship, claim 8 is rejected under 35 U.S.C. 112(a) for lack of enablement.
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 1-8 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.
Regarding claim 1, the claim recites a “method for determination of concentration of mutated form of mucin-1 protein in a biological sample and determination of position of its mutation,” followed by steps comprising adding the biological sample to an immobilized antibody against the mutated form of variable number tandem repeats (VNTR) of mutated frame-shifted variant of mucin-1 protein (MUC1fs), adding an antibody against the wild-type form of VNTR MUC1fs to “a portion of this mixture” and performing a quantitative analysis, and adding an antibody against the mutated form of VNTR MUC1fs to “the remainder of the mixture” and performing quantitative analysis. Claim 1 is indefinite because the relationship between the stated determinations in the introductory language and the positively recited assay steps is unclear. Specifically, although the claim identifies the method as being for both “determination of concentration” of mutated MUC1 and “determination of position of its mutation,” the body of the claim merely requires two quantitative analyses using different antibodies. The claim does not recite a step of determining the concentration of MUC1fs from either quantitative analysis, a step of determining the position of the mutation, or any relationship between the quantitative analyses and the stated determinations. The claim therefore does not make clear whether “determination of concentration” and “determination of position of its mutation” constitute required results of the claimed method, or instead merely identify an intended purpose for carrying out the recited assay steps. If the determinations are intended as affirmative limitations, it is further unclear what positively recited step or result constitutes the claimed determination of mutation position.
The uncertainty is particularly relevant to “determination of position of its mutation,” because the claim does not require comparing the two quantitative analyses, determining a ratio between the analyses, normalizing one analysis relative to the other, applying a calibration curve, determining respective lengths of wild-type and mutated VNTR regions, or otherwise processing either quantitative result to obtain positional information. The indefiniteness arises because the claim does not clearly establish whether determining mutation position is a required limitation at all and, if it is required, what recited act or result satisfies that requirement. For purposes of compact prosecution, the preamble will be interpreted broadly as requiring the method to provide quantitative information concerning MUC1fs concentration and information concerning the position of the mutation, without requiring an unrecited comparison, ratio, normalization, calibration procedure, exact positional coordinate, or particular positional classification. To the extent the introductory language merely expresses the intended purpose of the otherwise complete assay steps, the operative method limitations remain the positively recited capture and two quantitative antibody-analysis branches. Appropriate correction is required.
Claim 1 additionally recites adding the antibody against the wild-type form of VNTR MUC1fs to “a portion of this mixture,” followed by adding the antibody against the mutated form of VNTR MUC1fs to “the remainder of the mixture of the biological sample with the immobilized antibody against the mutated form of VNTR MUC1fs.” Although the preceding step recites adding the biological sample to the immobilized antibody, the claim does not expressly recite dividing, separating, or otherwise establishing a portion and remainder of the resulting material. Consequently, when the claim is considered together with the uncertainty concerning the two quantitative analyses, the language does not clearly define whether “a portion” and “the remainder” require physically separated aliquots of the sample/antibody mixture, sequential treatment of different portions of the same material, or some other partition of the material. For purposes of compact prosecution, the phrase “this mixture” will be interpreted as the material resulting from adding the biological sample to the immobilized antibody against the mutated form of VNTR MUC1fs. The phrase “a portion of this mixture” will be interpreted broadly as a portion or aliquot of that material that is contacted with an antibody against the wild-type form of VNTR MUC1fs, and the remainder of the mixture will be interpreted broadly as another portion of that material that is contacted with an antibody against the mutated form of VNTR MUC1fs. Appropriate correction is required.
Claims 2–8 depend from claim 1 and therefore incorporate the indefinite limitations of claim 1. Accordingly, claims 2–8 are rejected under 35 U.S.C. 112(b) at least by virtue of their dependency from claim 1.
Regarding claims 4-7, each claim recites “measurement of a change in absorbance of the solution.” However, neither claim 1 nor claims 4-7 previously introduces a solution to provide antecedent basis for “the solution.” Although claims 4-7 recite subsequent addition of a mixture of reactive dye and hydrogen peroxide, it is unclear whether the solution refers to that mixture, the biological-sample/antibody material recited in claim 1, the resulting assay mixture following addition of the reactive dye and hydrogen peroxide, or some other solution. Accordingly, the identity of the solution whose change in absorbance is measured is unclear, rendering claims 4-7 indefinite. For purposes of compact prosecution, the phrase “the solution” will be interpreted broadly as the assay mixture resulting after addition of the recited mixture of reactive dye and hydrogen peroxide, the absorbance of which is subsequently measured. Appropriate correction is required.
Regarding claim 8, the claim recites that, “for determination of the concentration of MUC1fs in the sample, determination of the VNTR length, and determination of the mutation position,” a length of the mutated region of VNTR MUC1fs is determined using an “appropriate calibration curve” obtained by analyzing a standard containing at least one epitope of the mutated form of VNTR MUC1fs, and a length of the wild-type region is determined using an “appropriate calibration curve” obtained by analyzing a standard containing at least one epitope of the wild-type form of VNTR MUC1fs. Claim 8 is indefinite because it expressly identifies three determinations—MUC1fs concentration, VNTR length, and mutation position—but positively recites only determination of the length of the mutated region and length of the wild-type region using respective calibration curves. The claim does not define what relationship between those two regional-length determinations constitutes the recited overall VNTR length or mutation position, nor does it specify how those regional-length determinations provide the recited concentration of MUC1fs. For example, the claim does not state that the wild-type and mutated region lengths are to be summed to determine VNTR length, that one of the regional lengths identifies the mutation position, or that any specified mathematical relationship between the two regional lengths is required.
For purposes of compact prosecution, the phrase “length of the mutated region of VNTR MUC1fs” will be interpreted broadly as a quantitative determination concerning the extent or length of the mutated VNTR region based on a calibration curve generated using a standard containing at least one epitope of the mutated form of VNTR MUC1fs. Correspondingly, the phrase “length of the wild-type region of VNTR MUC1fs” will be interpreted broadly as a quantitative determination concerning the extent or length of the wild-type VNTR region based on a calibration curve generated using a standard containing at least one epitope of the wild-type form. The phrase “VNTR length” will be interpreted broadly as length information concerning the VNTR, and “mutation position” will be interpreted broadly as positional information concerning the mutation within the VNTR. The claim is not interpreted as requiring an exact nucleotide coordinate, exact repeat number, or any particular unrecited mathematical operation for deriving mutation position. The phrase “concentration of MUC1fs” will be interpreted as a quantitative amount or concentration of MUC1fs resulting from the claimed assay. No particular mathematical relationship between concentration and VNTR-region length is imported into the claim beyond the limitations actually recited. Appropriate correction is required.
Additionally, the phrase “appropriate calibration curve” does not itself identify what makes a particular calibration curve appropriate for converting the quantitative antibody measurement into a determination of the length of a VNTR region. Although the claim specifies that the respective standards contain at least one epitope of the corresponding mutant or wild-type form, it does not expressly define the calibration variable or relationship by which the measured response corresponds to VNTR-region length. Accordingly, the scope of what calibration curve satisfies the claimed appropriate calibration curve is unclear. For purposes of compact prosecution, the phrase “appropriate calibration curve” will be interpreted as a calibration curve suitable for relating the corresponding assay measurement obtained from the specified standard to the claimed regional-length determination. This interpretation permits examination but does not resolve the uncertainty concerning what calibration relationship the claim affirmatively requires. Appropriate correction is required.
Ultimately, these interpretations are adopted solely to permit examination under compact prosecution and does not resolve or withdraw the foregoing §112(b) rejection.
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.
Claim 1 is rejected under 35 U.S.C. 103 as being unpatentable over Knaup et al. (Biallelic Expression of Mucin-1 in Autosomal Dominant Tubulointerstitial Kidney Disease: Implications for Nongenetic Disease Recognition. Journal of the American Society of Nephrology, Vol. 29, No. 9, September 2018) in view of Hey et al. (Transmembrane and Truncated (SEC) Isoforms of MUC1 in the Human Endometrium and Fallopian Tube. Reproductive Biology and Endocrinology. Vol. 1, No. 1, January 2003).
Regarding claim 1, for a method for determination of concentration of mutated form of mucin-1 protein in a biological sample and determination of position of its mutation, Knaup teaches immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique (Abstract, p. 2298). Knaup further teaches that MUC1 mutations occur within a domain of variable number of tandem repeats (VNTR), and that the resulting mucin 1 frameshift protein (MUC1-fs) is of variable length (depending on the number of repeats) (p. 2299). Regarding adding a biological sample to an immobilized antibody against the mutated form of variable number tandem repeats (VNTR) of mutated frame-shifted variant of mucin-1 protein (MUC1fs), Knaup teaches that polyclonal antibodies against MUC1-fs were generated using a recombinant peptide coded by one repeat of the frameshifted VNTR sequence as immunogen (p. 2301), and that the anti-MUC1fs sera specifically detect endogenous MUC1-fs by immunoblotting (p. 2303; Fig. 3). Regarding adding an antibody against the wild-type form of VNTR MUC1fs to a portion of this mixture, Knaup teaches that patient’s MUC1-fs will harbor wild-type and frameshifted repeats, and therefore both pAb3-fs and the wild-type VU4H5 antibodies are able to detect the identical protein (p. 2303; Fig. 3C). Knaup further physically divides patient-derived material for the respective antibody interrogations: the lanes of the membrane were cut into two halves, of which one half was incubated with pAb3-fs and the other one half was incubated with VU4H5 (p. 2303; Fig. 3D).
Knaup additionally explains why the wild-type VNTR antibody detects MUC1fs: the antibody directed against the native VNTR (VU4H5) will usually also detect the product from a mutated allele of any patient with ADTKD-MUC1, because the duplication mutation usually takes place after a succession of normal repeats, which can be immunodetected (p. 2301; Fig. 3A). Regarding adding an antibody against the mutated form of VNTR MUC1fs to the remainder of the mixture of the biological sample with the immobilized antibody against the mutated form of VNTR MUC1fs, Knaup teaches the mutant-VNTR antibody pAb3-fs and, as discussed above, expressly teaches separate treatment of the divided patient-derived material wherein one half was incubated with pAb3-fs while the other half received VU4H5 (p. 2303; Fig. 3D). Knaup further establishes that pAb3-fs and VU4H5 recognize the same MUC1fs protein containing the respective frameshifted and wild-type repeat regions.
Knaup itself supplies a particularly direct motivation to improve its detection format. Knaup explains that existing molecular analysis is technically demanding and very labor intensive, is available in only a restricted number of laboratories, and expressly states that it would, therefore, be beneficial to develop an easier, possibly nongenetic assay, which could be applied on a broader level (p. 2301). Knaup then develops MUC1fs antibodies toward that stated objective. Thus, Knaup gives the skilled artisan a reason to seek an established antibody-based MUC1 assay format capable of converting Knaup’s demonstrated MUC1fs antibody recognition into a practical quantitative assay.
However, Knaup does not teach immobilizing its mutant-VNTR MUC1fs antibody as a capture antibody, contacting that immobilized antibody with the biological sample, and quantitatively analyzing captured MUC1fs using the respective detector antibodies in a solid-phase assay. Knaup’s relevant experiments instead employ immunoblotting and immunohistochemistry.
Hey supplies these missing assay-format teachings. Regarding adding a biological sample to an immobilized antibody, Hey teaches a Dual determinant Enzyme Linked Immunosorbant Assay (ELISA) in which HPLC-purified BC2 (IgG) was employed as a capture antibody and was coated on 96 well plates and, after washing, 50 µl/well of sample applied for an overnight incubation (p. 5). Hey specifically teaches that BC2 recognizes the repeating epitope PDTRP within the MUC1 VNTR domain (p. 5). Thus, Hey establishes a solid-phase MUC1 capture assay directed to the VNTR domain. Regarding the use of different detector antibodies on captured MUC1, Hey teaches that a second anti-repeat McAb BC3 (IgM) was used as detector and, alternatively, McAb 7H10/5 (IgM) raised against MUC1/SEC specific peptide VSIGLSFPMLP was used as detector (p. 5). Hey therefore demonstrates that immobilized antibody-captured MUC1 can be interrogated using alternative antibodies directed to different MUC1 epitopes.
Regarding performing quantitative analysis, Hey teaches addition of a peroxidase-conjugated rabbit anti-mouse IgM antibody, followed by substrate reagent containing 0.02% hydrogen peroxide, and the reaction product was measured by absorbance at 405 nm in an automated plate reader (p. 5). Hey’s Figure 3 further identifies BC2 as the capture antibody, BC3 or 7H10/5 as alternative detector antibodies, and states that the vertical axis represents absorbance at 405 nm (p. 8, Fig. 3). Hey additionally reports that its conventional MUC1 ELISA (anti-VNTR capture and detector) confirmed the presence of variable amounts of MUC1 in all samples assayed (p. 7). Hey’s statement that quantitative comparisons between its different detectors are not possible (p. 7) does not defeat the rejection because claim 1, under the broadest reasonable interpretation applied here, requires quantitative analysis of the respective antibody interrogations but does not require a quantitative comparison between their signals.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Knaup’s MUC1fs immunodetection method by employing Hey’s solid-phase quantitative MUC1 immunoassay technique, including immobilizing Knaup’s MUC1fs-specific antibody as the capture antibody, contacting the immobilized antibody with a biological sample, and separately detecting the captured MUC1fs using Knaup’s wild-type-VNTR and mutant-VNTR antibodies with quantitative readout, because Knaup expressly identifies the need for an easier, nongenetic assay capable of broader application, while Hey provides precisely an established antibody-capture format for quantitatively assaying MUC1 using an immobilized anti-MUC1 capture antibody and alternative epitope-specific detector antibodies. This modification directly advances Knaup’s stated objective by replacing its blot-based antibody interrogation with a solid-phase quantitative MUC1 assay while retaining Knaup’s MUC1fs-specific antibody recognition. A person of ordinary skill would have had a reasonable expectation of success because Knaup experimentally establishes that pAb3-fs and VU4H5 both recognize the identical endogenous MUC1fs protein containing frameshifted and wild-type VNTR repeats and successfully applies those antibodies separately to divided patient-derived material, while Hey demonstrates that MUC1 can be captured by an immobilized antibody and thereafter detected using alternative MUC1 antibodies to generate measurable enzymatic signals. Thus, the proposed modification applies Hey’s demonstrated MUC1 immunoassay architecture to the MUC1fs antibody-antigen interactions already experimentally validated by Knaup.
Claim 2 is rejected under 103 as being unpatentable over Knaup et al. and Hey et al., as applied to claim 1 above, and further in view of Kirby et al. (Mutations Causing Medullary Cystic Kidney Disease Type 1 Lie in a Large VNTR in MUC1 Missed by Massively Parallel Sequencing. Nature Genetics. Vol. 45, No. 3, March 2013).
With respect to the teachings of Knaup and Hey, refer to the discussion above, which applies equally here. However, the combination does not expressly teach or specify the particular SEQ ID NO. 1 in the orientation recited by claim 2.
Kirby supplies the precise epitope. Kirby teaches that immunodetection of MUC1-fs was performed with custom-prepared rabbit antibodies (PA4 302) raised against the peptide SPRCHLGPGHQAGPGLHRPP, representing the predicted mutant VNTR unit (Methods, p. 305). Kirby further demonstrates actual immunodetection using those antibodies. Thus, Kirby expressly teaches an antibody against the mutant MUC1fs VNTR unit generated against the exact SEQ ID NO. 1 recited by claim 2. Kirby also reports that detection of bound primary antibody was achieved using either the EnVision+ Peroxidase Rabbit kit or System-HRP labeled Polymer Anti-mouse (Dako) for rabbit or mouse antibodies (Methods, p. 305) and separately describes immunofluorescence detection of PA4 302, establishing that the sequence-specific antibody was functional for immunodetection.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the Knaup-Hey assay by using Kirby’s MUC1fs antibody recognizing SPRCHLGPGHQAGPGLHRPP as the mutant-VNTR antibody because Kirby expressly identifies that peptide as the predicted mutant VNTR unit and demonstrates antibodies raised against that precise MUC1fs sequence for immunodetection. Kirby’s antibody performs the same mutant-MUC1fs recognition required by Knaup’s assay, against the same disease-associated frameshift product. A person of ordinary skill would have had a reasonable expectation of success because Kirby experimentally used antibodies raised against that exact mutant VNTR peptide for MUC1fs immunodetection, while Knaup independently demonstrates that frameshift-VNTR-directed antibodies specifically recognize endogenous MUC1fs.
Claim 3 is rejected under 103 as being unpatentable over Knaup et al. and Hey et al., as applied to claim 1 above, and further in view of Cao et al. (Mucins (MUC1 and MUC3) of Gastrointestinal and Breast Epithelia Reveal Different and Heterogeneous Tumor-Associated Aberrations in Glycosylation. Journal of Histochemistry & Cytochemistry. Vol. 45. No. 11, November 1997).
With respect to the teachings of Knaup and Hey, refer to the discussion above, which applies equally here. However, the combination does not expressly teach or specify the particular SEQ ID NO. 2 in the orientation recited by claim 3.
Cao teaches the precise wild-type MUC1 VNTR epitope. Cao states that the antibody A76-A/C7 (IgG1κ) was developed in this laboratory and that it recognizes the peptide epitope APDTRP of the MUC1 tandem repeat (p. 1549). Cao further applies its anti-MUC1 monoclonal antibodies to MUC1 protein detection in tissue (pp. 1549–1551). Thus, Cao expressly teaches a functional anti-MUC1 antibody recognizing the exact APDTRP sequence required by claim 3.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the Knaup-Hey assay by employing Cao’s A76-A/C7 antibody, which recognizes APDTRP of the MUC1 tandem repeat, as the antibody against the wild-type VNTR region because Knaup requires recognition of the normal VNTR repeats retained in MUC1fs and Cao expressly provides an anti-MUC1 antibody directed to a defined peptide epitope within that tandem repeat. The modification therefore implements Knaup’s wild-type-VNTR interrogation using Cao’s expressly characterized VNTR-specific antibody rather than introducing an unrelated assay function. A person of ordinary skill would have had a reasonable expectation of success because Cao experimentally establishes A76-A/C7 as an anti-MUC1 antibody recognizing APDTRP of the tandem repeat, while Knaup establishes that normal VNTR repeats remain on MUC1fs and are accessible to wild-type VNTR antibody recognition.
Claims 4 and 5 are rejected under 103 as being unpatentable over Knaup et al. and Hey et al., as applied to claim 1 above, and further in view of Yamashita et al. (EP 3088896 A1).
With respect to the teachings of Knaup and Hey, refer to the discussion above, which applies equally here. However, the combination does not expressly teach or specify one of the alternatives recited in claims 4 and 5.
Regarding claim 4, Yamashita teaches precisely the pertinent direct fluorescent anti-MUC1 detection technique. Yamashita teaches that a ternary sandwich immunocomplex composed of an immobilized anti-MUC1 antibody, Tn-MUC1 and an anti-MUC1 antibody bound with a fluorescent substance (fluorescently labeled anti-MUC1 antibody) is formed ([0056], p. 9). Yamashita further teaches that the fluorescently labeled anti-MUC1 antibody can be prepared by binding a desired fluorescent substance to an anti-MUC1 antibody and that a known fluorescent dye that is capable of emitting an appropriate fluorescence in Surface Plasmon-field enhanced Fluorescence Spectroscopy (SPFS) can be used ([0058], p. 9). Particularly relevant to applying the technique to Knaup, Yamashita expressly lists VU-4H5—the same wild-type MUC1 VNTR antibody used by Knaup—among commercially available anti-MUC1 monoclonal antibodies ([0054], p. 9). Regarding subsequent measurement of a change in fluorescence value, Yamashita teaches that by measuring the intensity of the fluorescence emitted from the fluorescent substance, the measurement target can be quantified based on the measured value ([0044], p. 8). Yamashita explains that this value can be converted into concentration using a known-concentration standard and that SPFS is sufficiently sensitive to determine targets present at an extremely low concentration ([0044], p. 8). Yamashita also experimentally prepares an Alexa Fluor 647-labeled anti-MUC1 antibody ([0096], p. 13) and measures light intensity corresponding to fluorescence in the signal measurement step ([0105]–[0108]).
Regarding claim 5, as discussed above, Yamashita further teaches that the fluorescently labeled anti-MUC1 antibody can be prepared by binding a desired fluorescent substance to an anti-MUC1 antibody ([0058], p. 9), and that fluorescence intensity can be measured to quantify the target ([0044], p. 8). Thus, Yamashita supplies the same direct fluorescent-labeling and quantitative fluorescence-readout technique for application to the mutant-VNTR anti-MUC1fs detector antibody recited by claim 5.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the Knaup-Hey quantitative assay by fluorescently labeling the respective wild-type-VNTR antibody of claim 4 and mutant-VNTR MUC1fs antibody of claim 5 and quantifying their respective binding by fluorescence according to Yamashita because Yamashita expressly teaches direct fluorescent labeling of anti-MUC1 antibodies for quantitative MUC1 sandwich immunoassays and teaches that fluorescence intensity permits quantitative determination of the target, including determination at extremely low target concentrations. For claim 4, Yamashita additionally identifies VU-4H5, the same wild-type VNTR antibody employed by Knaup, among available anti-MUC1 antibodies. The modification therefore changes the signal-generation mechanism without requiring a change in the antigen-recognition function on which Knaup’s MUC1fs detection depends. A person of ordinary skill would have had a reasonable expectation of success because Knaup establishes that both its wild-type-VNTR and mutant-VNTR antibodies recognize MUC1fs, while Yamashita demonstrates that anti-MUC1 antibodies can be conjugated to a fluorescent substance and used as detector antibodies to quantitatively measure MUC1; thus, applying Yamashita’s demonstrated fluorescent labeling/readout technique to either of Knaup’s respective MUC1-reactive detector antibodies would predictably preserve its antigen-recognition function while providing the claimed quantitative fluorescence signal.
Claims 6 and 7 are rejected under 103 as being unpatentable over Knaup et al. and Hey et al., as applied to claim 1 above, and further in view of Linden et al. (MUC1 Limits Helicobacter Pylori Infection Both by Steric Hindrance and by Acting as a Releasable Decoy. PLoS Pathogens, Vol. 5, No. 10, October 2009).
With respect to the teachings of Knaup and Hey, refer to the discussion above, which applies equally here. However, the combination does not expressly teach or specify one of the alternatives recited in claims 6 and 7.
Regarding claims 6 and 7, Hey teaches the secondary-antibody detection architecture applicable to either detector antibody: a peroxidase-conjugated rabbit anti-mouse IgM antibody, followed by 2 mM 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing 0.02% hydrogen peroxide, after which the reaction product was measured by absorbance at 405 nm in an automated plate reader (p. 5). Thus, Hey teaches converting MUC1 detector-antibody binding into a quantitative signal through a secondary-antibody/peroxidase/reactive-substrate/hydrogen-peroxide/absorbane sequence, but does not expressly identify the peroxidase as HRP.
Linden supplies the expressly recited HRP secondary-antibody species in a closely analogous MUC1 immunoassay. Linden teaches that the BC2 and BC3 antibodies are against the extracellular domain of MUC1 and react with a VNTR repeat epitope that is exposed even in fully glycosylated MUC1 (p. 11). Linden further teaches coating microtitre plates with the BC2 antibody, incubating the wells with cell lysate or gastric juice extract, and thereafter incubating the captured material with the anti-MUC1 primary antibody (clone BC3) (p. 11). Linden then expressly teaches that the plates were washed and incubated with HRP-conjugated anti-mouse IgM, and that HRP activity was determined using 2,2′-Azinobis(3-ethylbenzothiazoline)-6-sulphonic acid as a substrate by measuring absorbance at 405 nm (p. 11). Linden therefore expressly demonstrates HRP-conjugated anti-mouse IgM as the secondary-antibody signal-generating reagent in a MUC1 antibody assay employing an immobilized MUC1 capture antibody and an anti-MUC1 detector antibody. Linden’s Figure 1 further confirms that MUC1 from cell lysate and human gastric juice was captured using a microtitre plate coated with BC2 and detected with BC3 (p. 2). Linden also reports quantitative MUC1 determinations using the BC2/BC3 double-determinant ELISA, including concentrations of free MUC1 in conditioned medium determined by ELISA.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the Knaup-Hey quantitative MUC1fs assay by employing Linden’s HRP-conjugated anti-mouse IgM as the peroxidase-conjugated secondary antibody used to detect the respective wild-type-VNTR antibody of claim 6 and mutant-VNTR MUC1fs antibody of claim 7. Hey already teaches the pertinent secondary-antibody detection sequence of a peroxidase-conjugated anti-mouse IgM antibody followed by ABTS containing hydrogen peroxide and quantitative absorbance measurement at 405 nm, while Linden expressly demonstrates that HRP is a suitable particular peroxidase for an anti-mouse IgM secondary antibody in a closely analogous MUC1 BC2/BC3 capture-and-detection ELISA using ABTS and absorbance measurement at 405 nm. Thus, Linden provides a direct teaching to employ HRP as the particular peroxidase in Hey’s secondary-antibody detection arrangement, thereby providing the expressly recited HRP enzyme while retaining Hey’s established quantitative MUC1 immunoassay architecture. A person of ordinary skill would have had a reasonable expectation of success because Linden experimentally demonstrates HRP-conjugated anti-mouse IgM for detecting an anti-MUC1 primary antibody in a MUC1 capture assay, while Hey independently demonstrates the same type of anti-mouse IgM secondary-antibody architecture together with ABTS, hydrogen peroxide, and absorbance measurement at 405 nm. Accordingly, employing Linden’s expressly taught HRP conjugate as the particular peroxidase conjugate in Hey’s detection arrangement would have predictably generated the quantitative enzymatic signal for either of Knaup’s respective MUC1fs-reactive detector antibodies.
Conclusion
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/E.O./Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 October 5, 2026
