DETAILED ACTION
Request for reconsideration of the application filed on 02/12/2026, is acknowledged. No amendment was made to the claims. Claims 1-15 are pending in the application and are considered on merits.
In response to reconsideration, the examiner maintains rejections over prior art established in the previous Office action.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lippok et al (Biophysical Journal, 2013, IDS) (Lippok) in view of Torres et al. (Clinical Chemistry, 2012, IDS) (Torres) and Varghese et al. (Alalytica Chimica Acta, 2008) (Varghese).
Regarding claim 1, Lippok teaches a method for obtaining information on von Willebrand factor (VWF) (abstract), comprising the following steps:
denaturing, with urea, VWF contained in a biological sample (page 1210, par 5);
fluorescently labeling the denatured VWF using a capturing agent that comprises a fluorescent substance (eGFP) and binds to the denatured VWF (page 1210, par 5); and
obtaining information on the size of the fluorescently-labeled VWF by
fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy (page 1210, par 4).
Lippok does not specifically teach that wherein when the information is obtained by fluorescence correlation spectroscopy, the capturing agent comprises a polyclonal antibody, or a plurality of monoclonal antibodies or aptamers that bind to epitopes different from each other; and
wherein when the information is obtained by fluorescence cross-correlation spectroscopy, the capturing agent comprises a first capturing agent that comprises a first fluorescent substance and binds to the denatured VWF, and a second capturing agent that comprises a second fluorescent substance and binds to the denatured VWF,
wherein the second fluorescent substance is a fluorescent substance having a maximum fluorescence emission in a wavelength range different from that of the first fluorescent substance, and
wherein the first capturing agent that comprises the first fluorescent substance
and binds to the denatured VWF is a polyclonal antibody that comprises the first
fluorescent substance, or a plurality of monoclonal antibodies or aptamers that each
comprise the first fluorescent substance and bind to epitopes different from each other.
However, Torres teaches that that wherein when the information is obtained by fluorescence correlation spectroscopy, the capturing agent comprises monoclonal antibody (page 1012, par 4).
The use of polyclonal antibodies or multiple monoclonal antibodies targeting different epitopes of the same antigen was well known in immunoassays and protein detection techniques at the time of the invention. A person of ordinary skill in the art would have reasonably understood that multiple antibodies recognizing different epitopes may be used to improve binding efficiency and detection reliability. Therefore, selecting a polyclonal antibody or a plurality of monoclonal antibodies as the capturing agent would have been an obvious design choice in view of the known properties of antibody binding. and
Varghese teaches that wherein when the information is obtained by fluorescence cross-correlation spectroscopy, the capturing agent comprises a first capturing agent that comprises a first fluorescent substance and binds to the target protein, and a second capturing agent that comprises a second fluorescent substance and binds to the target protein (page 104, par 3-4),
wherein the second fluorescent substance is a fluorescent substance having a maximum fluorescence emission in a wavelength range different from that of the first fluorescent substance (page 104, par 3), and
wherein the first capturing agent that comprises the first fluorescent substance
and binds to the denatured VWF is a monoclonal antibody that
comprise the first fluorescent substance and bind to an epitope (page 104, par 3).
wherein the second fluorescent substance is a fluorescent substance having a maximum fluorescence emission in a wavelength range different from that of the first fluorescent substance (page 104, par 3).
Lippok demonstrates that FCS reliably measures VWF multimer size under urea-denaturing conditions.
Torres confirms the practicality of fluorescently labeled monoclonal anti-VWF antibodies in FCS.
Varghese extends FCS to dual-color FCCS for analyzing protein interactions and denaturation.
A person of ordinary skill in biophysical assay design would have recognized that combining the immuno-FCS labeling of Torres with the dual-color FCCS configuration of Varghese would yield enhanced signal discrimination and multiplex capability in the VWF analysis framework of Lippok.
Therefore, it would have been obvious to one of ordinary skill in the art to modify Lippok’s FCS method for VWF size analysis to employ the fluorescent monoclonal antibody labeling of Torres and the dual-color FCCS setup of Varghese to obtain size and interaction information on denatured VWF using spectrally distinct capturing agents, in order to obtain specific VWF multi-site binding, that yield enhanced signal discrimination and multiplex capability in the VWF analysis framework of Lippok. The combination yields predictable results.
Regarding 7, Lippok teaches a method for preparing a measurement sample for use in fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy (abstract), comprising the following steps:
denaturing, with urea, von Willebrand factor (VWF) contained in a biological sample (page 1210, par 4); and
fluorescently labeling the denatured VWF using a capturing agent that comprises a fluorescent substance and binds to the denatured VWF (page 1210, par 4).
Lippok does not specifically teach that
wherein when the VWF fluorescently labeled with the capturing agent is used for measurement by fluorescence correlation spectroscopy, the capturing agent comprises a polyclonal antibody, or a plurality of monoclonal antibodies or aptamers that bind to epitopes different from each other; and
wherein when the VWF fluorescently labeled with the capturing agent is used for measurement by fluorescence cross-correlation spectroscopy, the capturing agent comprises a first capturing agent that comprises a first fluorescent substance and binds to the denatured VWF, and a second capturing agent that comprises a second fluorescent substance and binds to the denatured VWF,
wherein the second fluorescent substance is a fluorescent substance having a maximum fluorescence emission in a wavelength range different from that of the first fluorescent substance, and
wherein the first capturing agent that comprises the first fluorescent substance and binds to the denatured VWF is a polyclonal antibody that comprises the first fluorescent substance, or a plurality of monoclonal antibodies or aptamers that each comprise the first fluorescent substance and bind to epitopes different from each other.
However, Torres teaches that that wherein when the information is obtained by fluorescence correlation spectroscopy, the capturing agent comprises monoclonal antibody (page 1012, par 4).
The use of polyclonal antibodies or multiple monoclonal antibodies targeting different epitopes of the same antigen was well known in immunoassays and protein detection techniques at the time of the invention. A person of ordinary skill in the art would have reasonably understood that multiple antibodies recognizing different epitopes may be used to improve binding efficiency and detection reliability. Therefore, selecting a polyclonal antibody or a plurality of monoclonal antibodies as the capturing agent would have been an obvious design choice in view of the known properties of antibody binding. and
Varghese teaches that wherein when the information is obtained by fluorescence cross-correlation spectroscopy, the capturing agent comprises a first capturing agent that comprises a first fluorescent substance and binds to the target protein, and a second capturing agent that comprises a second fluorescent substance and binds to the target protein (page 104, par 3-4),
wherein the second fluorescent substance is a fluorescent substance having a maximum fluorescence emission in a wavelength range different from that of the first fluorescent substance (page 104, par 3), and
wherein the first capturing agent that comprises the first fluorescent substance and binds to the denatured VWF is a monoclonal antibody that comprise the first fluorescent substance and bind to an epitope (page 104, par 3).
wherein the second fluorescent substance is a fluorescent substance having a maximum fluorescence emission in a wavelength range different from that of the first fluorescent substance (page 104, par 3).
Lippok demonstrates that FCS reliably measures VWF multimer size under urea-denaturing conditions.
Torres confirms the practicality of fluorescently labeled monoclonal anti-VWF antibodies in FCS.
Varghese extends FCS to dual-color FCCS for analyzing protein interactions and denaturation.
A person of ordinary skill in biophysical assay design would have recognized that combining the immuno-FCS labeling of Torres with the dual-color FCCS configuration of Varghese would yield enhanced signal discrimination and multiplex capability in the VWF analysis framework of Lippok.
Therefore, it would have been obvious to one of ordinary skill in the art to modify Lippok’s FCS method for VWF size analysis to employ the fluorescent monoclonal antibody labeling of Torres and the dual-color FCCS setup of Varghese to obtain size and interaction information on denatured VWF using spectrally distinct capturing agents, in order to obtain specific VWF multi-site binding, that yield enhanced signal discrimination and multiplex capability in the VWF analysis framework of Lippok. The combination yields predictable results.
Regarding 2, Lippok teaches that wherein obtaining the information comprises obtaining a diffusion time of the fluorescently-labeled VWF by fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy (page 1210, par 3).
Regarding 3, Lippok teaches that wherein the information is the diffusion time, or a value obtained based on the diffusion time (page 1210, par 3).
Regarding 4 and 8, Torres and Varghese teach that wherein when the information is obtained by fluorescence cross-correlation spectroscopy, the second capturing agent that comprises the second fluorescent substance and binds to the denatured VWF is a plurality of monoclonal antibodies that each comprise the second fluorescent substance and bind to epitopes different from each other (Torres, page 1012, par 4; Varghese, page 104, par 3-4).
Regarding 5 and 9, Lippok teaches that wherein the denaturing is carried out in the presence of urea at a concentration of not less than 0.5 M and not more than 1.75 M (1.5 M) (page 1210, par 4).
Regarding 6 and 10, It would have been obvious to one of ordinary skill in the art to optimize the concentration of urea by routine experimentation.
Regarding claim 11, Lippok discloses a reagent kit for use in the method according to claim 1, comprising urea and a capturing agent that comprises a fluorescent substance and binds to urea-denatured von Willebrand factor (VWF) (page 1210, par 4). Torres and Varghese disclose that wherein the capturing agent comprises a plurality of monoclonal antibodies that bind to epitopes different from each other (Torres, page 1012, par 4; Varghese, page 104, par 3-4).
Regarding claim 12, Lippok discloses that wherein the urea is contained in a urea reagent solution, and wherein the concentration of urea in the urea reagent solution is not less than 1 M and mot more than 8 M (page 1210, par 4).
Regarding claim 13, Lippok in view of Torres and Varghese fairly suggest to one of ordinary skill in the art a reagent kit for use in the method according to claim 1 using fluorescence cross correlation spectroscopy, comprising urea, a first capturing agent that comprises a first fluorescent substance and binds to urea-denatured VWF, and a second capturing agent that comprises a second fluorescent substance and binds to the urea-denatured VWF, wherein the second fluorescent substance is a fluorescent substance having a maximum absorption in a wavelength region different from that of the first fluorescent substance, and wherein the first capturing agent that comprises the first fluorescent substance and binds to the urea-denatured VWF is a polyclonal antibody that comprises the first fluorescent substance, or a plurality of monoclonal antibodies or aptamers that each comprise the first fluorescent substance and bind to epitopes different from each other.
Regarding claim 14, Torres and Varghese disclose and fairly suggest that wherein the second capturing agent that comprises the second fluorescent substance and binds to the urea-denatured VWF is a plurality of monoclonal antibodies that each comprise the second fluorescent substance and bind to epitopes different from each other (Torres, page 1012, par 4; Varghese, page 104, par 3-4).
Regarding claim 15, Lippok discloses that wherein the urea is contained in a urea reagent solution, and wherein the concentration of urea in the urea reagent solution is not less than 1 M and mot more than 8 M (page 1210, par 4).
Response to Arguments
Applicant's arguments filed 02/12/2026 have been fully considered but they are not persuasive.
Applicant argues that the cited references fail to recognize the technical problem of “concentration dependency” in fluorescence correlation spectroscopy (FCS) measurements of VWF and that the claimed configuration of urea denaturation and multi-epitope capturing agents provides a unique solution. However, the rejection under 35 U.S.C. §103 does not rely on recognition of the same problem identified by Applicant.
It is well established that a reference need not recognize the same problem addressed by the applicant in order to render the claimed invention obvious. See In re Kahn, 441 F.3d 977, 988 (Fed. Cir. 2006) and KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 419-420 (2007). If the prior art provides teachings that would have suggested the claimed elements to a person of ordinary skill in the art, the combination may still be obvious regardless of whether the references explicitly identify the same technical issue described in the specification.
Lippok teaches measuring the size distribution of von Willebrand factor (VWF) multimers using fluorescence correlation spectroscopy and further teaches performing the analysis in the presence of urea to stretch or partially denature the VWF molecules. Thus, Lippok already establishes the fundamental framework of analyzing VWF using FCS under urea denaturing conditions.
Torres teaches the use of fluorescently labeled monoclonal anti-VWF antibodies for detection of VWF by fluorescence correlation spectroscopy. Specifically, Torres describes incubating plasma samples with TRITC-labeled monoclonal anti-VWF antibodies prior to FCS measurement. Accordingly, Torres demonstrates that antibody-based fluorescent capturing agents for VWF were well known in FCS analysis of VWF.
Applicant argues that Torres teaches only a single monoclonal antibody and therefore does not suggest the use of polyclonal antibodies or multiple monoclonal antibodies recognizing different epitopes. However, the use of polyclonal antibodies or multiple monoclonal antibodies targeting different epitopes of the same antigen was well known in immunoassays and protein detection techniques at the time of the invention. A person of ordinary skill in the art would have reasonably understood that multiple antibodies recognizing different epitopes may be used to improve binding efficiency and detection reliability. Therefore, selecting a polyclonal antibody or a plurality of monoclonal antibodies as the capturing agent would have been an obvious design choice in view of the known properties of antibody binding.
Applicant further argues that Varghese teaches away from the claimed subject matter because Varghese reports that urea can inhibit binding of IgG and anti-IgG at high concentrations. This argument is not persuasive. Varghese merely demonstrates that certain protein–protein interactions may be weakened at sufficiently high urea concentrations. However, Varghese does not discourage the use of urea generally in fluorescence correlation spectroscopy systems. Rather, Varghese studies the effects of denaturation conditions on protein interactions using dual-color fluorescence correlation spectroscopy. A reference teaches away only when it criticizes, discredits, or otherwise discourages the claimed solution. See In re Fulton, 391 F.3d 1195, 1201 (Fed. Cir. 2004). Varghese does not criticize or discourage the use of urea in fluorescence correlation spectroscopy experiments; instead, it analyzes protein interactions under denaturing conditions. Accordingly, Varghese does not teach away from the combination relied upon in the rejection.
Applicant also argues that the presently claimed invention demonstrates unexpected stability of diffusion time at low antigen concentrations when polyclonal antibodies or antibody cocktails are used. However, the evidence presented in the specification compares a single monoclonal antibody with a polyclonal antibody configuration without demonstrating that such results would have been unexpected to one of ordinary skill in the art. The use of multivalent or multi-epitope antibody binding to enhance antigen capture and improve measurement stability is consistent with the known behavior of antibody-antigen systems and would have been expected by those skilled in immunochemical assay design.
With respect to claim 6, Applicant argues that the claimed stepwise urea concentration differs from the constant urea concentration used in Lippok. However, adjusting reagent concentrations during different stages of sample preparation and measurement is a routine optimization within the ordinary skill of the art. Lippok already teaches the use of urea to control VWF conformation for FCS measurement. A person of ordinary skill in the art would have understood that the concentration of denaturant could be varied between preparation and measurement steps to balance denaturation and binding stability depending on the experimental requirements. Such adjustment represents routine optimization of known assay parameters rather than a patentable distinction.
Therefore, the combination of Lippok, Torres, and Varghese continues to teach or suggest the claimed method. Lippok provides the framework of FCS analysis of VWF under urea conditions, Torres teaches fluorescent anti-VWF antibodies used for FCS detection, and Varghese teaches dual-color fluorescence correlation spectroscopy using distinct fluorescent labels for interacting proteins. A person of ordinary skill in the art would have been motivated to combine these teachings to obtain information on VWF size and interactions using fluorescence correlation techniques with antibody-based labeling.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to XIAOYUN R XU, Ph. D. whose telephone number is (571)270-5560. The examiner can normally be reached M-F 8am-5pm.
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/XIAOYUN R XU, Ph.D./ Primary Examiner, Art Unit 1797