DETAILED ACTION
The amendment and RCE filed on 06/17/2026 have been entered and fully considered. Claims 5-6 and 9-10 are canceled. Claims 1-4, 7-8 and 11-15 are pending, of which claims 1 and 7 are amended.
Response to Amendment
In response to amendment, the examiner maintains rejection over the 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) 1-4, 7-8 and 11-15 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, wherein the denaturing is carried out in the presence of urea at a first concentration of not less than 0.5 M and not more than 1.75 M (1.5 M urea) (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 the fluorescent labeling is carried out in the presence of urea at a second concentration of not less than 0.2 M and not more than 1 M, and wherein the second concentration is less than the first concentration;
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), and
urea concentration affects antibody-antigen binding interactions and specifically demonstrates that higher concentrations of urea can weaken or inhibit such binding interactions (Fig. 6, page 108, par 1).
Lippok demonstrates that FCS reliably measures VWF multimer size under urea-denaturing conditions (page 1210, par 5).
Torres confirms the practicality of fluorescently labeled monoclonal anti-VWF antibodies in FCS (page 1012, par 4).
Varghese extends FCS to dual-color FCCS for analyzing protein interactions and denaturation (page 104, par 304), and further teaches that urea concentration affects antibody-antigen binding interactions and specifically demonstrates that higher concentrations of urea can weaken or inhibit such binding interactions (Fig. 6, page 108, par 1).
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.
Lippok teaches the use of urea during fluorescence correlation spectroscopy analysis of VWF to alter VWF conformation and facilitate size measurements (page 1210, par 4). Torres teaches fluorescently labeled anti-VWF antibodies for FCS analysis of VWF (page 1012, par 4). Varghese teaches that urea concentration affects antibody-antigen binding interactions and specifically demonstrates that higher concentrations of urea can weaken or inhibit such binding interactions (Fig. 6, page 108, par 1). Thus, the combined references establish both (1) the desirability of using urea to denature or unfold proteins and (2) the known effect that excessive urea concentrations can adversely affect antibody binding.
In view of these teachings, it would have been obvious to one of ordinary skill in the art to employ a higher urea concentration during an initial denaturation step and a lower urea concentration during a subsequent antibody-labeling step in order to balance protein unfolding with maintenance of adequate antibody binding. Such adjustment merely involves optimizing a result-effective variable. The concentration of urea was recognized in the art as affecting both protein conformation and antibody-antigen interaction, and discovering workable or optimal concentrations for different stages of the assay would have been within the routine skill of the ordinary artisan. See In re Aller, 220 F.2d 454, 456 (CCPA 1955).
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, wherein the denaturing is carried out in the presence of urea at a first concentration of not less than 0.5 M and not more than 1.75 M (1.5M urea) (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 the fluorescent labeling is carried out in the presence of urea at a second concentration of not less than 0.2 M and not more than 1 M, and wherein the second concentration is less than the first concentration;
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 urea concentration affects antibody-antigen binding interactions and specifically demonstrates that higher concentrations of urea can weaken or inhibit such binding interactions (page 108, par 1) 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 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 (page 1210, par 4).
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.
In view of these teachings, it would have been obvious to one of ordinary skill in the art to employ a higher urea concentration during an initial denaturation step and a lower urea concentration during a subsequent antibody-labeling step in order to balance protein unfolding with maintenance of adequate antibody binding. Such adjustment merely involves optimizing a result-effective variable. The concentration of urea was recognized in the art as affecting both protein conformation and antibody-antigen interaction, and discovering workable or optimal concentrations for different stages of the assay would have been within the routine skill of the ordinary artisan. See In re Aller, 220 F.2d 454, 456 (CCPA 1955).
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.
Lippok teaches the use of urea during fluorescence correlation spectroscopy analysis of VWF to alter VWF conformation and facilitate size measurements (page 1210, par 4). Torres teaches fluorescently labeled anti-VWF antibodies for FCS analysis of VWF (page 1012, par 4). Varghese teaches that urea concentration affects antibody-antigen binding interactions and specifically demonstrates that higher concentrations of urea can weaken or inhibit such binding interactions (Fig. 6, page 108, par 1). Thus, the combined references establish both (1) the desirability of using urea to denature or unfold proteins and (2) the known effect that excessive urea concentrations can adversely affect antibody binding.
In view of these teachings, it would have been obvious to one of ordinary skill in the art to employ a higher urea concentration during an initial denaturation step and a lower urea concentration during a subsequent antibody-labeling step in order to balance protein unfolding with maintenance of adequate antibody binding. Such adjustment merely involves optimizing a result-effective variable. The concentration of urea was recognized in the art as affecting both protein conformation and antibody-antigen interaction, and discovering workable or optimal concentrations for different stages of the assay would have been within the routine skill of the ordinary artisan. See In re Aller, 220 F.2d 454, 456 (CCPA 1955).
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 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 06/17/2026 have been fully considered but they are not persuasive.
Applicant argues that Lippok fails to disclose or suggest a process in which VWF is denatured at a first urea concentration and subsequently labeled at a lower urea concentration, and further argues that such a stepwise concentration adjustment is not routine optimization. Applicant's arguments have been considered but are not persuasive.
Lippok teaches the use of urea during fluorescence correlation spectroscopy analysis of VWF to alter VWF conformation and facilitate size measurements (abstract). Torres teaches fluorescently labeled anti-VWF antibodies for FCS analysis of VWF (page 1010). Varghese teaches that urea concentration affects antibody-antigen binding interactions and specifically demonstrates that higher concentrations of urea can weaken or inhibit such binding interactions (Fig. 6, page 108, par 1). Thus, the combined references establish both (1) the desirability of using urea to denature or unfold proteins and (2) the known effect that excessive urea concentrations can adversely affect antibody binding.
In view of these teachings, it would have been obvious to one of ordinary skill in the art to employ a higher urea concentration during an initial denaturation step and a lower urea concentration during a subsequent antibody-labeling step in order to balance protein unfolding with maintenance of adequate antibody binding. Such adjustment merely involves optimizing a result-effective variable. The concentration of urea was recognized in the art as affecting both protein conformation and antibody-antigen interaction, and discovering workable or optimal concentrations for different stages of the assay would have been within the routine skill of the ordinary artisan. See In re Aller, 220 F.2d 454, 456 (CCPA 1955).
Applicant further argues that Lippok teaches a constant concentration and therefore teaches away from changing concentration during the assay. However, a disclosure of one suitable operating condition does not teach away from other workable conditions absent criticism, discrediting, or discouragement of such alternatives. Lippok merely performs its measurements at a particular urea concentration and does not criticize, discredit, or otherwise discourage adjustment of urea concentration between assay stages. Accordingly, Lippok does not teach away from the claimed concentration relationship.
Applicant additionally argues that Varghese teaches away from the claimed subject matter because Varghese reports inhibition of antibody-antigen binding at elevated urea concentrations. This argument is not persuasive. Varghese does not discourage the use of urea; rather, Varghese informs the skilled artisan that antibody binding is affected by urea concentration (page 108, par 1). Such teaching would have suggested reducing urea concentration when stronger antibody binding is desired. Therefore, Varghese provides further motivation for employing a lower urea concentration during the labeling step than during the denaturation step.
Applicant's evidence of allegedly unexpected results is likewise not persuasive. The claims encompass broad concentration ranges and do not require the specific experimental conditions shown in the examples. Moreover, the reported improvement in binding stability is consistent with the expected result of reducing denaturant concentration during an antibody-binding step. Applicant has not provided evidence establishing that the asserted results would have been unexpected throughout the full scope of the claims or that the results are commensurate in scope with the claimed subject matter.
Accordingly, the rejection under 35 U.S.C. §103 is maintained.
Conclusion
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
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