DETAILED ACTION
Final Action
Notice of Pre-AIA or AIA Status
1. 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 Claims
2. Claims 1, 3-4, 6, 8, 14, 20, 22, 26, 30, 130, 149 and 166-167 as amended on 09/11/2025 are pending and under examination in this office action.
Priority
3. Acknowledgement is made of Applicants claim for priority to U.S. Provisional Patent Application Ser. No. 63/081,647, filed 22 September 2020.
Withdrawn Claim Objections
4. Objections to claims 1, 3-4, 8, 14, 20, 22, 26, 30 and 166 are withdrawn in view of amendment to the claims filed on 09/11/2025.
Claim Rejections - 35 USC § 112
5. Withdrawn rejection of claims 1, 3-4, 6, 8, 14, 20, 22, 26, 30, and 166 under 35 U.S.C. 112(b) in view of amendment to the claims filed on 09/11/2025.
6. Withdrawn rejection of claim 8 on the ground of insufficient antecedent basis in view of amendment to the claims filed on 09/11/2025.
Claim Rejections - 35 USC § 103 (Amended)
7. 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.
8. Claim 1 is rejected under 35 U.S.C. 103 as being unpatentable over Chou et al 2004 (published in Journal of Virological Methods 123 (2005) 41–48) and further in view of Bickerstaff et al 2007 (J Virol Methods, 2007 Jun;142(1-2):50-58), Molinos-Albert et al 2014 (Retrovirology. 2014 Jun 7; 11:44), Huang et al 2020 (published on 03 August 2020, Acta Pharmacologica Sinica (2020) 41:1141–1149), Horndler et al 2020 (published medRxiv preprint doi: doi.org,10.1101,2020.08.24.20180661; version posted August 25, 2020), Grzelak et al 2020 (Grzelak et al., Sci. Transl. Med. 12, eabc3103, p. 1-13), Huang et al 2020 (Acta Pharmacologica Sinica (2020) 41:1141–1149), and McKay et al 2020 (bioRxiv, preprint posted online on 04/25/2020), and as evidenced by Hatmal et al 2020 (Cells, 2020, 9, 2638, p. 1-37).
Claim 1: The instant claim 1 is interpreted as directed to a method of determining whether a biological fluid sample comprises an antibody that binds to a SARS CoV-2 coronaviral antigen, the method, inter alia, comprises a human cell expressing SARS CoV-2 coronaviral antigenic determinant fused to a fluorescent protein detectable domain wherein the SARS CoV-2 coronaviral antigen anchored in the cell membrane is expressed on the human cell surface and the fluorescent protein detectable domain is expressed inside the cell, and the fluorescent protein is fused to the SARS CoV-2 coronaviral antigen via membrane anchored protein, the antibody bound to SARS CoV-2 coronaviral antigen is detected by a fluorescent label conjugated secondary antibody.
Regarding claim 1: Chou et al 2004 is in the virology and SARS CoV art. Chou et al discloses a novel cell-based binding assay …… for SARS-CoV S protein and its cellular receptor. Chou discloses a plasmid construct comprising a SARS CoV S gene fused to EGFP (See, page 42 for methods column 2, para 2, page 44, figure 1-A and associated legends) wherein the SARS-CoV S protein gene encoding ectodomain is fused to EGFP (enhanced green fluorescent protein) encoding sequence through the transmembrane domain (TM) and cytoplasmic tail domain (CT) of SARS CoV S gene and forming a fusion DNA construct S-EGFP; the transmembrane and cytoplasmic domains of the S protein were retained in the construct for proper anchoring and orientation of the ectodomain on the surface of CHO cells, a full length S protein gene was used for extracellular cell surface expression and display of SARS-CoV S protein fused intracellular expressed EGFP, an extra octapeptide (TRDPPVAT) from the EGFP vector was generated at the junction, which should have limited effect on the transportation and expression of the fusion protein to arrive at fusion protein S-EGFP and it is inherent that membrane anchored protein comprises a lipid anchor; as well as the intracellular localization of a detectable domain EGFP; the expression of S-EGFP on cell surface was detected by using nonpermeable immunofluorescence assay involving use of SARS CoV patients convalescent serum, Rhodamine fluorescent labelled secondary antibody anti-human IgG , the binding of serum antibodies and it involved washing steps to remove or separate the labeled composition from unbound labeled secondary binding member (Rhodamine fluorescent labelled secondary antibody anti-human IgG) and read our was done using fluorescence microscope (See, page 43, column 2, Results; page 44 -45, section 3.3 immunofluorescence analysis, figure 3 legends, figures 1B, 2-3). A stable CHO cell clone, CHO-SG (SARS CoV- Spike protein and EGFP), expressing a relatively high level of S-EGFP, at the cytoplasmic membrane was selected (See, page 43, Results, column 2, para 1; page 44-45, figures 1B, 2-3). Non-permeable immunofluorescence staining was used identify cell surface expression of S-EGFP, CHO-SG cells were dislodged with 0.04% EDTA in PBS, fixed after incubating with primary antibody (patients’ serum) and secondary anti-human IgG conjugated with Rhodamine label (See, Chou, page 43, col 1, section 2.3 on Immunofluorescence analysis). Chou et al demonstrates detection of serum from a SARS convalescent patient (patients SARS CoV antibodies) bind to the cell surface expressed SARS CoV S protein/antigenic determinants and secondary anti-human IgG Rhodamine label conjugated fluorescent (secondary member) binds to the primary antibody (See, Chou, abstract, page 45, Figure 3 and legend). Huang et al 2020 discloses intracellular detectable domain FP of S protein (See, Huang et al, page 1143, column 2, last paragraph, fig 1-2) and the FP structure/location is disclosed in a form of intracellular EGFP by Chou et al as recited, supra (reference recited for clarity of the inventive concept).
Chou et al do not teach instant claim 1 limitation (a) (ii) a human cell displaying on surface an antigenic determinant of SARS CoV-2 and (c) a flow cytometry to assay antibodies in a sample.
Bickerstaff et al 2007 is in the art teaching the application of a flow cytometry for detection/quantification of antibody response to murine cytomegalovirus infection. The target cells Murine embryo fibroblast NIH/3T3 cells infected with green fluorescent protein labeled murine cytomegalovirus (GFP-MCMV) expresses envelope antigenic determinants on a cell surface to which the antibodies in sample binds, and dual color flow cytometry detects GFP expressed in cell and MCMV antibodies bound to viral envelope antigenic determinants on the surface of the infected cell (See, Bickerstaff et al 2007, abstract, p. 51 Methods, Results p. 52- 54 Figures 1-3, entire article).
Molinos-Albert et al 2014 is in the art dealing with application of flow cytometry for detection/quantification of antibody response to HIV in infected individuals. Molinos-Albert et al 2014 teaches that antibodies are induced against MPER region of the HIV-1 envelope glycoprotein gp41. Flow cytometry was used to quantify and characterize anti-MPER antibodies using a collection of mini gp41-derived proteins (antigenic determinants) expressed on the surface of human 293T cells and GFP protein expressed inside the cell (See, abstract, p. 9-12 Molinos-Albert et al 2014, Method, p.3 Figure 1 A and B; Figures 2-5, entire article).
Thus, both, Bickerstaff et al 2007 and Molinos-Albert et al 2014 teaches the inventive concept of claim 1 disclosing application of flow cytometry to determine antibodies bound to a viral antigen expressed and displayed on a cell surface and a GFP protein marker inside a cell expressing viral antigen on the surface of the cell. Molinos-Albert et al 2014 specifically discloses a HIV gp41-TM-GFP construct to express gp41 on a cell surface for use in a method for determining or quantification of anti-HIV antibodies in a sample using gp41 expressed on 293-T cell surface, a transmembrane domain and a GFP fluorescent protein as a marker inside the 293-T cell expressing the gp41 antigen on a 293-T cell surface (See, Molinos-Albert et al 2014, p.3, Fig 1, A and associated legends).
Chou et al, Bickerstaff et al and Molinos-Albert et al do not teach claim 1 (a) (ii) limitation wherein the coronaviral antigen is derived from SARS-CoV-2.
Grzelak et al 2020 teaches a comparison of four serological assays for detecting anti–SARS-CoV-2 antibodies in human serum samples from different populations including SARS CoV-2 S protein flow cytometry assay that recognized the S protein expressed at the cell surface using flow cytometry (See, abstract; p.6 Fig 2, p.8-10 Figs 3-5; Methods p.10 col 2 S-Flow assay continued... p. 11 col 1 para 1). Grzelak et al 2020 do not teach intracellular expression of fluorescent protein GFP fused to the SARS CoV-2 protein displayed on a human cell.
Horndler et al teaches instant added limitations claim 1 (a) (ii) limitation wherein the coronaviral antigen is derived from SARS-CoV-2. Horndler et al also additionally teaches flow cytometry for determining SARS-CoV-2 antibodies in a sample by disclosing blood and mucosal fluids including total saliva, human serum samples from coronavirus infected patients (Horndler teaches claim 26 limitation). Horndler et al teaches the steps of the flow cytometry method including binding of the antibodies from serum / plasma to the SARS CoV-2 S glycoprotein expressed on the Jurkat cell surface as Jurkat-S, washing steps with PBS-BSA buffer and light centrifugation (spun) to eliminate the excess of antibody (See, Horndler et al, page 1 for abstract, pages 11-12 for material and methods for cells, human sera samples, flow cytometry).
McKay et al 2020 is in the art and teaches a method and steps of flow cytometry for detection of SARS CoV-2 S antibody bound to the S protein expressed on surface of human cells HEK293T/17 cells (See, page 10).
Huang et al 2020 teaches SARS CoV-2 spike protein structural domains (See, Huang et al 2020, p. 1143 Fig 2-a, entire article).
Hatmal et al 2020 in a review provides evidence on similarity of comprehensive structural and molecular comparison of Spike proteins of SARS-CoV-2, and SARS-CoV and MERS-CoV (See, Hatmal et al 2020, p. 4, Fig 1B).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the prior art teachings of Chou et al 2004 and incorporate the teachings of Bickerstaff et al 2007, Molinos-Albert et al 2014 and McKay et al 2020 on application of flow cytometry method for determining viral antibodies in a sample, incorporate additional teachings on viral antigen expressed on surface of human 293 T cells and further incorporate teachings of SARS CoV and SARS CoV-2 Spike protein structural organization as taught by Huang et al, Hatmal et al 2020, Grzelak et al 2020 and Horndler et al 2020 on SARS CoV-2 antigenic determinants on S protein to arrive at the invention of claim 1. One of the ordinary skills in the art would have been motivated develop a human cell expressing SARS CoV-2 antigenic determinants on cell surface with an intra-cellular GFP based flow cytometry to assure specific detection and quantification of SARS CoV-2 antibodies in body fluids or serum or plasma of subjects. The human cell surface expression of the SARS CoV-2 antigenic determinant would facilitate access to native confirmation of coronaviral antigenic determinants for use as an antigen in the flow cytometry assay (See, Chou et al, abstract). One of the motivations would be to monitor transfection efficiency and thereby expression of SARS CoV-2 antigen by assessing intracellular expression of GFP (See, Chou, page 44 fig 1; page 45 fig 3). In addition, the post-translational modification pattern of the expressed SARS CoV-2 protein would be identical/highly similar in a human cell as that of natural infection and expression of SARS CoV-2 antigen (S protein) on a cell of infected human. The SARS CoV (taught by Chou et al) and SARS CoV-2 (taught by Horndler et al) belongs to the genus Betacoronavirus of the family Coronaviridae that share a structural organization as taught by Huang et al and Hatmal et al 2020 (an evidence) and therefore, there would have been a reasonable expectation of success to replace SARS CoV S protein antigenic determinants in the expression construct with SARS CoV-2 S protein and antigenic determinants to arrive at the invention of claim 1 given the combined prior art teachings of Chou et al 2004, Bickerstaff et al 2007, Molinos-Albert et al 2014, Huang et al 2020, Grzelak et al 2020, Horndler et al 2020, Huang et al, and Hatmal et al (an evidence) as recited supra.
9. Claims 3-4, 6, 8, 14, 20, 22, 26, 30 and 166 are rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of Chou et al 2004 (published in Journal of Virological Methods 123 (2005) 41–48), Bickerstaff et al 2007 (J Virol Methods, 2007 Jun;142(1-2):50-58), Molinos-Albert et al 2014 (Retrovirology. 2014 Jun 7; 11:44), McKay et al 2020 (bioRxiv, preprint posted online on 04/25/2020), Huang et al 2020 (published on 03 August 2020, Acta Pharmacologica Sinica (2020) 41:1141–1149), Grzelak et al 2020 (Grzelak et al., Sci. Transl. Med. 12, eabc3103, p. 1-13), Horndler et al 2020 (published medRxiv preprint doi: doi.org,10.1101,2020.08.24.20180661; version posted August 25, 2020), Huang et al 2020 (Acta Pharmacologica Sinica (2020) 41:1141–1149) and further in view of Mori et al 2020 (Sci Rep 10, 4798, p. 1-12) as evidenced by Hatmal et al 2020 (Cells, 2020, 9, 2638, p. 1-37).
The combined teachings of Chou et al 2004, Bickerstaff et al 2007, Molinos-Albert et al 2014, Huang et al 2020, Grzelak et al 2020, Horndler et al 2020, Huang et al 2020 and Hatmal et al 2020 (evidence) teaches claim 1 as recited supra.
Claims 3-4, 6, 8, 14, 20, 22, 26, 30 and 166: The combined prior art teachings as applied to claim 1 above further teaches added limitations of claims 3-4, 6, 8, 14, 20, 22, 26, 30 and 166 as recited below:
Chou et al is in the SARS CoV art and teaches added limitation of instant claims 3, wherein the CHO cell membrane anchored protein comprises a transmembrane protein (See, Chou et al, p. 43, col 2, results, p. 44 Fig. 1 A S-EGFP construct). Huang et al 2020, Hatmal et al 2020 (evidence) and Horndler et al teaches SARS CoV-2 and spike protein antigenic determinants. Molinos-Albert et al is in virology HIV art and teaches transient and stable expression in human embryonic kidney HEK-293 cells used to express gp41 on the cell surface using a gp41-TM-GFP construct (See, Molinos-Albert et al, p.3, fig 1; p.9 col 2, para 2).
Claim 4. Chou et al is in the SARS CoV art teaches added limitation of instant claims 4, wherein the CHO cell membrane anchored protein comprises a lipid anchor (See, Chou, p. 43, col 2, results). Huang et al 2020 and Hatmal et al 2020 (evidence) and Horndler et al teaches SARS CoV-2 and spike protein antigenic determinants. Molinos-Albert et al is in virology HIV art and teaches transient and stable expression in human embryonic kidney HEK-293 cells used to express gp41 on the cell surface using a gp41-TM-GFP construct (See, Molinos-Albert et al, p.3, fig 1; p.9 col 2, para 2).
Claim 6. Chou et al is in the SARS CoV art teaches added limitation of instant claim 6, wherein the detectible domain is present inside the CHO cell by disclosing the intracellular localization of a detectable domain EGFP (See, Chou, p. 43, col 2, results). Huang et al 2020 and Hatmal et al 2020 (evidence)and Horndler et al teaches SARS CoV-2 and spike protein antigenic determinants. Molinos-Albert et al is in virology HIV art and teaches transient and stable expression in human embryonic kidney HEK-293 cells used to express gp41 on the cell surface using a gp41-TM-GFP construct. The GFP is present inside the cell (See, Molinos-Albert et al, p.3, fig 1; p.9 col 2, para 2).
Claim 8. Chou et al is in the SARS CoV art and teaches added imitation of instant claim 8, wherein the CHO mammalian cell comprises an expression construct encoding the antigenic determinant of the SARS CoV coronaviral antigen (See, Chou, page 43, Results, column 2, para 1; page 44-45, figures 1B, 2-3). Huang et al 2020 and Hatmal et al 2020 (evidence) and Horndler et al teaches SARS CoV-2 and spike protein antigenic determinants. Molinos-Albert et al is in virology HIV art and teaches transient and stable expression in human embryonic kidney HEK-293 cells used to express gp41 on the cell surface using a gp41-TM-GFP construct, wherein the expressed GFP is present inside the HEK-293 cells (See, Molinos-Albert et al, p.3, fig 1; p.9 col 2, para 2).
McKay et al 2020 is in the art and teaches a method and steps of flow cytometry for detection of SARS CoV-2 S antibody bound to the S protein expressed on surface of HEK293T/17 cells (See, page 10).
Claim 14. Chou et al is in the SARS CoV art and teaches added limitation of instant claims 14, wherein the coronaviral antigen comprises a spike protein (See, Chou, abstract, fig 1 and legends). Huang et al 2020 and Hatmal et al 2020 (evidence)and Horndler et al teaches SARS CoV-2 and spike protein antigenic determinants. Molinos-Albert et al is in virology HIV art and teaches transient and stable expression in human embryonic kidney HEK-293 cells used to express gp41 on the cell surface using a gp41-TM-GFP construct. The GFP is present inside the cell (See, Molinos-Albert et al, p.3, fig 1; p.9 col 2, para 2).
Claim 166: The method of Claim 1, wherein the human cell is a HEK293 cell or a derivative thereof. Molinos-Albert et al is in virology HIV art and teaches added limitation of claim 166 by disclosing transient and stable expression in human embryonic kidney HEK-293 cells used to express gp41 on the cell surface using a gp41-TM-GFP construct. Mori et al 2020 teaches high transfection efficiency of the human embryonic kidney cell line HEK293 by measuring the target protein level in cells surviving after high-efficient, multicopy introduction of plasmid DNA by which the target protein is highly expressed under a cytomegalovirus promoter (See, p. 1, introduction para 2; p. 3 fig 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the invention of claim 1 with additional teachings of the prior arts that are applied to render obvious claim 1 to arrive at the inventions of claims 3-4, 6, 8, 14, and 166. One of the ordinary skills in the art would have been motivated to develop a human cell or stable human cell line expressing SARS CoV-2 antigenic determinants on a cell surface with an intra-cellular GFP to practice a method of flow cytometry to assure specific detection and quantification of SARS CoV-2 antibodies in body fluids or serum or plasma of a human or a subject. The human cell surface expression of the SARS CoV-2 antigenic determinant would facilitate access to native confirmation of coronaviral antigenic determinants for use as an antigen in the flow cytometry assay (See, Chou et al, abstract). One of the motivations would be to monitor transfection efficiency and thereby expression of SARS CoV-2 antigen by assessing intracellular expression of GFP that is fused to the SARS CoV-2 antigenic determinant TM domain to ensure the specificity of antibody in a sample bound to the cell surface displayed SARS CoV-2 antigenic determinant(s) (See, Chou, page 44 fig 1; page 45 fig 3). In addition, the post-translational modification pattern of the expressed SARS CoV-2 protein would be identical/highly similar in a human cell as that of natural infection and expression of SARS CoV-2 antigen (S protein) on a cell of infected human and high transfection efficiency of HEK293 cell line (See, Mori et al, p. 1, introduction, para 2). The invention reciting the method of the flow cytometry assay would be safer than use of live SARS CoV-2 virus infected human cells as a source of cell surface displayed antigenic determinant(s). There would have been a reasonable expectation of success to arrive at the inventions of claims 3-4, 6, 8, 14, and 166 given the combined prior art teachings applied to the claims as recited supra.
Claims 20 and 22: Horndler et al teaches added limitation of instant claims 20 and 22, wherein the labeled secondary binding member comprises an antibody or binding fragment thereof (claim 20 limitation); and wherein the labeled secondary binding member comprises a fluorescent label (claim 22 limitation) by disclosing a secondary antibody mouse anti-human IgG1 Fc-PE used in coronaviral antibody detection flowcytometry (See, Horndler et al, page 12, method-flow cytometry). It is obvious to one of ordinary skill in the art to use a fluorescent reporter PE labelled secondary antibody mouse anti-human IgG1 Fc-PE because the samples are human sera comprising human IgG to which the labelled secondary antibody mouse anti-human IgG1 Fc-PE will bind after wash step following binding of patient’s antibodies to the S antigen and flowcytometry requires a fluorescent reporter label to detect signal. The motivation to use a labeled secondary binding member (secondary antibody) comprising fluorescent label is that flowcytometry requires a fluorescent reporter labeled secondary antibody to detect and differentiate SARS CoV S protein /coronaviral antigen binding by patients’ serum sample specific binding to generate assay readout signal that would be different wavelength than GFP fluorescent signal. There would have been a reasonable expectation of success to arrive at the invention of claims 20 and 22 given the combined prior art teachings applied to render obvious the claim 20 and 22, as recited supra.
Claim 26: Horndler et al teaches instant added limitation of instant claim 26, wherein the sample comprises a biological fluid. (See, Chou et al, abstract, and Horndler et al, page 1 for abstract, pages 11-12 for material and methods for cells, human sera samples, flow cytometry).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the invention of claim 1 with additional teachings of the prior art Chou et al to arrive at the inventions of claim 26 on use of biological fluid sample – serum, plasma because antibodies induced to SARS CoV virus infection are present in the biological fluid of infected human or a subject. It is routine in infectious diseases to use biological fluid as a source of disease marker antibody and used for detection variety of assay including flowcytometry assay approach disclosed by Horndler et al. One of the ordinary skills in the art would have been motivated to develop a a cell-based flowcytometry assay suitable for detection of SARS CoV-2 S coronaviral antibodies in body fluids or plasma of a human or a subject to detect and quantify antibody response and antibodies that bind to native confirmation of coronaviral antigenic determinants (See, Chou et al, abstract). There would have been a reasonable expectation of success to arrive at the invention of claim 26 given the combined prior art teachings applied to render obvious the claim 26.
Claims 30: Horndler et al teaches added limitation of instant claim 30, wherein the method further comprises determining an amount of the antibody present in the sample that binds to the coronaviral antigen.
Horndler et al teaches the added limitation of instant claim 30, wherein the method further comprises determining an amount of the antibody present in the sample that binds to the coronaviral antigen (See, Horndler, methods and results). Horndler teaches by disclosing the Jurkat-S cells expressing SARS CoV2 S and EGFR were treated with plasma or biological fluid sample , cells were washed with PBS and then treated with labeled secondary fluorescent conjugated antibody and after incubation to allow binding, was washed and subjected for flowcytometry assay reading and data analysis to determine positive and negative sample for SARS CoV-2 antibodies (See, Horndler, page 12, flow cytometry and page 5-10 results and discussion , legends for figures 2-4 and associated figures).. The assay data on MFI of positive plasma samples with SARS CoV2 S and EGFR protein, MFI of negative plasma samples with SARS CoV2 S and EGFR protein was analyzed to compute anti-S flowcytometry MFI and anti-S flowcytometry (ratio) to determine positive and negative cutoff for the samples. (See, figure 1-e); the flow cytometry assay for presence of a signal from the labelled secondary binding member to determine whether the sample comprises the antibody is shown in figures 2-4 (See, legends for figures 2-4 and associated figures). The Jurkat-S cells were analyzed using a flow cytometry (FC) assay, where staining with an anti-EGFR monoclonal antibody detects and quantitates expression of the huEGFRt construct alongside detection and quantitation of anti-S protein antibodies within sera from SARS-CoV2 infected blood donors. Figure 1e shows Jurkat-S cells stained with anti-EGFR mAb and either a serum sample taken from an individual before the COVID-19 pandemics (pre-COVID-19 serum) or serum taken from an asymptomatic donor (donor #15) determined positive with multiple assays including anti-S protein flow cytometry (Table 1, see last 2 columns for results on anti-S Flow cytometry MF). Serum from donor #15 could strongly detect the S protein expressed on Jurkat-S cells whereas pre-COVID-19 serum did not, proving this method to be valid for capture and detection of anti-Spike antibodies present in sera from SARS-CoV2-exposed individuals (See, Horndler et al, pages 5-10 for results and discussion). The EGFR serves a normalizing role and allows to accurately quantify the presence of anti-S immunoglobulins (antibodies) by calculating a ratio of the mean fluorescence intensities obtained by double-staining with the sera and anti-EGFR specific monoclonal antibody. The method allows to detect SARS CoV-2 S protein antibodies in a sample from individuals that are immune to SARS CoV-2 (Horndler teaches claim 30 limitation). The method can be used in a multiplexed format to simultaneously measure all anti-S human immunoglobulin isotypes in blood and mucosal fluids including total saliva samples (See, Horndler et al, abstract, Material and methods for cells, human sera samples, flow cytometry). The DNA construct comprising SARS CoV-2 spike protein gene and human EGFR genes used to produce Jurkat T cell line that stably express the full-length native spike “S” protein of SARS-CoV-2 and a truncated form of the human EGFR, (See, figure 1-a); expression of S protein on the surface/plasma membrane of Jurkat-S cells assessed by surface biotinylation (See figure 1-b) (Hondler teaches instant claims 13 limitation).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the instant claimed inventions to analyze the flow cytometry data obtained from positive and negative control serum or plasma samples and patients serum or plasma sample and analyze the data to determine samples that are positive and negative to identify the subjects /patients having coronaviral antibodies due to infection or vaccination (See, Horndler, for flowcytometry data analysis results and discussion). One of the ordinary skills in the art would have been motivated to incorporate Horndler et al teachings on flowcytometry data analysis to modify the combined prior art teachings applied to the claim 1 to determine the amount of the antibodies present in the sample that binds to the SARS CoV-2 antigenic determinants. There would have been a reasonable expectation of success to arrive at the invention of claim 30 given the combined prior art teachings as applied to claim 30 and recited supra.
Claim 166: The method of claim 1, wherein the human cell is a HEK293 cell or a derivative thereof.
Molinos-Albert et al is in virology HIV art and teaches transient and stable expression in human embryonic kidney HEK-293 cells used to express gp41 on the cell surface using a gp41-TM-GFP construct (See, Molinos-Albert et al, p.3, fig 1; p.9 col 2, para 2).
Mori et al 2020 teaches high transfection efficiency of the human embryonic kidney cell line HEK293 by measuring the target protein level in cells surviving after high-efficient, multicopy introduction of plasmid DNA by which the target protein is highly expressed under a cytomegalovirus promoter (See, p. 1, introduction para 2; p. 3 fig 1).
McKay et al 2020 is in the art and teaches a method and steps of flow cytometry for detection of SARS CoV-2 S antibody bound to the S protein expressed on surface of human cells HEK293T/17 cells (See, page 10).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the invention of claim 1 with additional teachings of Molinos-Albert et al on HIV gp41-TM-GFP construct design teaching the surface display of gp41 antigenic determinants as recited supra and Mori et al and McKay et al teachings that HEK 293 cells has high transfectability and incorporate these teachings to arrive at the invention of claim 160 claiming use of the human cell, HEK293 cell for surface display expression of SARS CoV-2 S antigenic determinants with TM and fused GFP domain for an intra-cellular expression of detectable fluorescent GFP protein. One of the ordinary skills in the art would have been motivated to use a highly transfectable and well characterized human HEK293 cell line for surface display expression of SARS CoV-2 antigenic determinants and to have similar post-translational modifications in the expressed antigen as that of a naturally infected human cell displaying SARS CoV-2 S protein on a human cell surface (See, Mori et al, p. 1, introduction, para 2). The human cell surface expression of the SARS CoV-2 antigenic determinant would facilitate access to native confirmation of coronaviral antigenic determinants for use as an antigen in the flow cytometry assay (See, Chou et al, abstract). The developed flow cytometry assay would be safer than use of live SARS CoV-2 virus infected cell displaying the viral antigenic determinants on the cell surface. There would have been a reasonable expectation of success to arrive at the inventions of claim 166 given the combined prior art teachings applied to the claim as recited supra.
10. Claims 130, 149 and 167 over combined teachings of Chou et al 2004 (published in Journal of Virological Methods 123 (2005) 41–48), Bickerstaff et al 2007 (J Virol Methods, 2007 Jun;142(1-2):50-58), Molinos-Albert et al 2014 (Retrovirology. 2014 Jun 7; 11:44), Huang et al 2020 (published on 03 August 2020, Acta Pharmacologica Sinica (2020) 41:1141–1149), Grzelak et al 2020 (Grzelak et al., Sci. Transl. Med. 12, eabc3103, p. 1-13), Horndler et al 2020 (published medRxiv preprint doi: doi.org,10.1101,2020.08.24.20180661; version posted August 25, 2020), Huang et al 2020 (Acta Pharmacologica Sinica (2020) 41:1141–1149), Mori et al 2020 (Sci Rep 10, 4798, p. 1-12), evidenced by Hatmal et al 2020 (Cells, 2020, 9, 2638, p. 1-37) and further in view of Glencross 2011 (US7892551B2 published 02/22/2011).
Claims 130, 149 and 167: The claims 130, 149 and 167 are directed to a kit comprising: a human cell displaying on a surface thereof an antigenic determinant of a coronaviral antigen, wherein the antigenic determinant is a component of a membrane anchored protein, wherein the coronaviral antigen is derived from SARS-CoV-2, wherein the membrane anchored protein comprises a fluorescent protein detectible domain; and a labeled secondary binding member that binds to an antibody that specifically binds to the antigenic determinant (claim 130 limitation); wherein the labeled secondary binding member comprises an antibody or binding fragment thereof (claim 149 limitation); wherein the human cell is a HEK293 cell or a derivative thereof (claim 149 limitation).
The combined teachings of Chou et al 2004, Bickerstaff et al 2007, Molinos-Albert et al 2014, Huang et al 2020, Grzelak et al 2020, Horndler et al 2020, Huang et al 2020, Mori et al 2020, evidenced by Hatmal et al 2020 that rendered obvious claims 1, 3-4, 6, 8, 14, 20, 22, 26, 30 and 166 as recited supra are incorporated here in its entirety and the combined teachings discloses all limitations of claims 130, 149 and 167 except the added limitation a kit.
Glencross 2011 (US7892551B2) teaches the concept of packaging components into a kit and is well known and routine in the art. For example, Tsimikas et al. teaches a kit including antibodies, wherein the antibodies consist of anti-CD4 and anti-CD45 antibodies for use in enumerating the number of CD4+ cells in a sample and are conjugated to a label for detection with flow cytometry, which further includes one or more reagents selected from the group consisting of a red cell lysating agent, a stabilizer, a fixative, control cells, and bead reagents. The kit further includes instructions for performing a method of enumerating the number of CD4+ cells in a cell sample (See, Glencross 2011, US7892551B2, claims 1-3, abstract and the prior art in entireity).
Glencross 2011 (US7892551B2) teaches that the concept of packaging reagents into kit for flow cytometry based quantitative diagnostic assay is known.
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 invention of claims 1, 3-4, 6, 8, 14, 20, 22, 26, 30 and 166 with additional teachings of Glencross 2011 (US7892551B2) on concept of packaging reagents into kit for flow cytometry to arrive at the invention of claims 130, 149 and 167. One of the ordinary skills in the art would have been motivated to package the claimed invention of the SARS CoV-2 antibody determination flow cytometry-based assay comprising the required reagents as recited supra in the kit format and package for logistic purpose and commercial success. There would have been a reasonable expectation of success to arrive at the inventions of claims 130, 149 and 167 given the combined prior art teachings applied to the claimed inventions as recited supra.
According to MPEP § 2112.01(III), “Where the only difference between a prior art product and a claimed product is printed matter that is not functionally related to the product, the content of the printed matter will not distinguish the claimed product from the prior art. In re Ngai, ** > 367 F.3d 1336, 1339, 70 USPQ2d 1862, 1864 (Fed. Cir. 2004) < (Claim at issue was a kit requiring instructions and a buffer agent. The Federal Circuit held that the claim was anticipated by a prior art reference that taught a kit that included instructions and a buffer agent, even though the content of the instructions differed.). See also In re Gulack, 703 F.2d 1381, 1385-86, 217 USPQ 401, 404 (Fed. Cir. 1983) ("Where the printed matter is not functionally related to the substrate, the printed matter will not distinguish the invention from the prior art in terms of patentability….[T]he critical question is whether there exists any new and unobvious functional relationship between the printed matter and the substrate).
Response to Arguments
11. Applicant’s arguments filed on 09/11/2025 with respect to claims 1, 3-4, 6, 8, 14, 20, 22, 26, 30, 130, 149 and 166-16 have been considered but was not found persuasive in view of amended rejection.
In view of applicant’s amendment of the claim 1 and 8 filed on 09/11/2025, upon further search and consideration, McKay et al 2020 was applied as a prior art for to the rejection of the claims 1, 3-4, 6, 8, 14, 20, 22, 26, 30, 130, 149 and 166-167 and the amended 35 USC 103 rejection is recited supra.
Applicant’s arguments: Applicant’s main argument is that the applied prior arts do not render obvious the pending claims 1, 3-4, 6, 8, 14, 20, 22, 26, 30, 130, 149 and 166-167 under 35 USC 103.
In Response: The inventive concept expression of SARS-CoV-2 S protein on the surface of human cell surface, HEK293 cells or derivative cells 293T cells and expression of a GFP protein inside the cell wherein SARS-CoV-2 S protein is fused to the GFP is rendered obvious by Chou et al by expression of SARS-CoV S protein on the CHO cell surface and EGFP protein fused to S protein of SARS-CoV and expressed inside the CHO cells as recited in the office action supra. Chou et al has provided motivation to the applicant for a human cell surface expression of S protein of SARS-CoV-2 that is fused to the GFP and expressed inside the human cells HEK293 cells or derivative cells 293T cells and further use the SARS-CoV-2 S fused GFP expressing cells for serology of SARS CoV-2 S antibody in serum or plasma. However, flow-cytometry is a routine technique in the art and is rendered obvious by the prior arts as applied to the claims and recited supra. The differences in structure and sequence of SARS CoV and SARS-COV-2 S protein does not make the claimed invention novel because the inventive concept is taught by Chou et al. Optimization of cell surface expression SARS-CoV-2 S protein fused to GFP based on combining the prior arts knowledge and skills of the ordinary is routine in the art for a motivation of commercial success (See, MPEP 2143, I, Examples of rationale A-G).
Applicant’s arguments filed on 09/11/2025 with respect to the amended claims 1, 3-4, 6, 8, 14, 20, 22, 26, 30, 130, 149 and 166-16 have been considered but were not found persuasive. The rejection of the claims 1, 3-4, 6, 8, 14, 20, 22, 26, 30, 130, 149 and 166-16 as recited supra in this office action is maintained.
Conclusion
12. No claim is allowed.
13. Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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.
14. Any inquiry concerning this communication or earlier communications from the examiner should be directed to SAMADHAN J JADHAO whose telephone number is (703)756-1223. The examiner can normally be reached M-F 8:00-5:00.
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/SAMADHAN JAISING JADHAO/Examiner, Art Unit 1672
/BENNETT M CELSA/Quality Assurance Specialist, Art Unit 1600
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