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 .
Priority
The present application was filed on 08/18/2022. Acknowledgment is made of the present application as a proper National Stage (371) entry of PCT/US2021/070105, filed on 02/01/2021. This application claims benefit of U.S. Provisional Patent Application 62/985,069 filed on 03/04/2020.
Election/Restrictions
Applicant affirms the election of:
a labeling agent encapsulated by a liposome for the amplifying reagent;
a biotin and streptavidin for the small molecules and the corresponding linkers.
However, Claims 8 and 14-16 are amended to depend, directly or indirectly, from claim 5. Accordingly, claims 1-5, 8-12, and 14-18 read on the elected species, so claims 8-12, 14-18 are rejoined with claims 1-5 for examination. While claim 19 depends on claim 14, claim 19 reads on the non-elected species the carrier protein, so it is not rejoined for examination.
Objection/Rejection status
The rejection of claim 2 under 35 USC 102 is withdrawn in view of the amendment of the claim.
The rejections of claims 1 and 3-5 under 35 USC 103 are withdrawn in view of the amendment of the claim.
New ground rejection is made in view of the amendment of the claims.
Claim status
Claims 1-19 are pending. Claims 6-7 and 13 are withdrawn from further consideration by the examiner, 37 CFR 1.142(b), as being drawn to a non-elected invention.
Claims 1-5, 8-12 and 14-18 are examined herein.
Claim Objections
Claim 1 is objected to because of the following informalities: missing --a-- in step b line 1 after “for”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 1-5, 8-12, and 14-18 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.
Claim 1, part b, lines 2-3, recites “the conjugate reagent bound to the linker reagent bound to the amplifying reagents bound to the linker reagents”. It is unclear if all the linkers are used to connect the conjugate reagent and the amplifying reagents or if one linker connects the conjugate reagent and the amplifying reagents and the other linkers only bound to the amplifying reagents but not the conjugate reagent.
Claims 4-5, 8-12, and 14-18 depend on a multiple dependent claim 3, and they are also multiple dependent claims because they refer to more than one claim previously set forth, e.g., the method (in claim 1) or kit (in claim 2) of claim 3. Claims 4-5, 8-12, and 14-18 are improper because a multiple dependent claim should refer to other claims in the alternative only and cannot depend on any other multiple dependent claims. See MPEP § 608.01(n). Claims 4-5, 8-12, and 14-18 are vague and indefinite because the metes and bounds of the claims cannot be determined because it is unknown what other limitations the claims inherit from their parent claim(s). See MPEP 608.01(n)
Claim 17 recites “a concentration ranging from at least … to at least ….” The term “at least” renders the claim indefinite because the required concentration of the encapsulated AE is unclear. It is unclear if the concentration is from 1x10-8 to 1x10-6 or at least 1x10-8 or at least 1x10-6.
Claim Rejections - 35 USC § 103
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.
Claims 1-5 and 8-11 are rejected under 35 U.S.C. 103 as being unpatentable over Peretz et al. (US20170131269) in view of Liu et al. (Nano-biotinylated liposome-based immunoassay for the ultrasensitive detection of protein biomarker in urine, Talanta 179 (2018) 472–477).
Regarding claims 1-2, Peretz discloses a method of detecting an analyte in a sample using a biotin-avidin amplifying system, wherein multiple avidin linker reagents and multiple amplifying reagents are used. The method comprises: combining, in a medium, the sample with a conjugate reagent, linker reagents, amplifying reagents, and, optionally, a capture binding partner for the analyte.
(See Fig.1A and par.6 teaching: incubating the sample with a surface that is coated with a binding agent that binds to the analyte; incubating the surface with the same or a different binding agent of the analyte that is labeled with biotin, incubating the surface with streptavidin; without removing the streptavidin, further incubating the surface with a labeled biotin (e.g., biotin-fluorophore). The teaching means that:
a conjugate reagent is a binding agent of the analyte that is labeled with biotin,
linker reagents are streptavidin molecules,
amplifying reagents are labeled biotin molecules (e.g., biotin-fluorophore or biotinylated PE),
a capture binding partner for the analyte is a binding agent that binds to the analyte.)
The method also comprises examining the medium for a bound analyte, the bound analyte comprising the analyte bound to the conjugate reagent bound to the linker reagent bound to the amplifying reagents bound to the linker reagents (see Fig.1A showing: the analyte bound to biotinylated antibody bound to the first streptavidin bound to biotinylated PE bound to the second streptavidin; and par.6 teaching: “detecting and/or quantitating the label and thereby detecting and/or quantitating the analyte”);
Fig.1A excerpted from Peretz
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wherein
the conjugate reagent comprises a detection binding partner for the analyte and a first small molecule (see Fig.1A and par.6 teaching: a conjugate reagent comprises a binding agent of the analyte (e.g., antibody for analyte) and a first small molecule (e.g., biotin));
each of the amplifying reagents comprises a second small molecule on its surface (see Fig.1A and par.6 teaching: the amplifying reagents are fluorophores and the second small molecules are biotin);
each of the linker reagents comprises a binding partner for the first small molecule and the second small molecule (see Fig.1A and par.6 teaching: the linker reagents are streptavidin that can bind to the first small molecule biotin and the second small molecule biotin).
Peretz teaches that the avidin-biotin amplification system is used widely in immunoassay because the system takes advantage of the multiple biotin-binding sites in each avidin or streptavidin molecule, thereby improving the sensitivity, allowing increased detection range, using fewer primary antibodies and antigen standards, and reducing the costs of the assay (see pars.4-5). Peretz provides a detecting immunoassay wherein the analyte is detected by using a conjugate reagent, multiple linker reagents, multiple amplifying reagents and a capture binding partner for the analyte.
Peretz also teaches a kit for detecting an analyte corresponding to the method of detecting an analyte in a sample. The kit comprises the reagents used in the method (see par.12 and par.64), e.g., a conjugate reagent (see par.12, par.64 and Fig.1: teaching a detection antibodies labeled with biotin); linker reagents (see par.12, par.64 and Fig.1: streptavidin); amplifying reagent (see par.64 and Fig.1: phycoerythrin labeled biotin); and optionally, a capture binding partner (see par.64: one or more binding agents, analyte-specific antibody). Peretz teaches that kits offering the streptavidin or avidin and biotinylated enzymes at optimized concentrations are commercially available, so that one can form the complex by simply mixing the reagents (see par.5).
Peretz does not teach the amplifying reagent comprises a labeling agent encapsulated by a liposome.
Liu teaches that a liposome-based immunoassay using signaling molecule-loaded liposomes whose surfaces have been tagged with the protein of interest have been developed for a variety of assays, e.g., a biotinylated liposome system (see page 472 col.2 par.2). This teaching encompasses the amplifying reagent comprising a labeling agent (i.e., signaling molecule) encapsulated by a liposome.
Liu also teaches that the biotinylated liposome system is easy to prepare, and shows stable performance. Moreover, the signal can be further amplified by an avidin–biotin bridge (see 472 col.2 par.2). Compared with conventional ELISA, liposome-encapsulated signals can produce 500–1000-fold signal enhancement (see 472 col.2 par.1).
Liu teaches, in Figure 1, a method of detecting an analyte in a sample using the biotinylated liposome system comprising: a biotinylated antibody (i.e., a conjugated reagent), an avidin (i.e., a linker reagent), a biotinylated liposome (i.e., an amplifying reagent).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the amplifying reagent (e.g., biotinylated PE) in the method and kit taught by Peretz with the amplifying reagent comprising a signaling molecule encapsulated by a biotinylated liposome taught by Liu because the amplifying reagents in Liu and Peretz are functionally equivalent in terms of generating a signal for immunoassay.
A person of ordinary skill in the art would have been motivated to use the biotinylated liposome system of Liu because it is easy to prepare and shows stable performance. Moreover, it produces 500–1000-fold signal enhancement compared to conventional immunoassay (see Liu see 472 col.2 par.2). One having an ordinary skill in the art would have had a reasonable expectation of success in combining Peretz and Liu because they are directed to the immunoassay where the labeling agents and the analyte detector are conjugated by using biotin-avidin bond. Moreover, the biotinylated liposomes of Liu can be used in an avidin-biotin amplification system of Peretz (see Liu 472 col.2 par.2 teaching that the signal can be further amplified by an avidin–biotin bridge).
Regarding claim 3, Peretz and Liu teach the method or kit of detecting an analyte in the sample as in claims 1 and 2. Peretz teaches that the detection binding partner of the conjugate reagent for the analyte comprises an antibody that specifically binds the analyte (see par.6 and 22 teaching: biotinylated binding agent that binds to the analyte include, but are not limited to, an antibody, an antibody fragment etc.)
Regarding claims 4-5, Peretz and Liu teach the method or kit of detecting an analyte in the sample as in claims 1 and 2. Peretz teaches that the first and second small molecule comprise biotin (see Fig.1A and par.6). Accordingly, the binding partner of biotin is avidin based molecule, e.g., streptavidin or neutravidin (see Fig.1A and par.6).
Regarding claims 8-9, Peretz and Liu teach the method or kit of detecting an analyte in the sample as in claims 1 and 2. Peretz discloses that the capture binding partner for the analyte further comprises a support (see par.6 and Fig.1 teaching a surface that is coated with a binding agent that binds to the analyte). The support can be any of a variety of surface, such as a magnetically responsive bead, or a microtiter plate well (see par.7).
Regarding claim 10, Peretz and Liu teach the method or kit of detecting an analyte in the sample as in claims 1 and 2. Peretz discloses that the method further comprises a washing step prior to the step of examining the medium for bound analyte (see par.6).
Regarding claim 11, Peretz and Liu teach the method or kit of detecting an analyte in the sample as in claims 1 and 2. Claim 11 recites that the conjugate reagent of the method or kit of claim 9 further comprises a labeling agent.
Peretz and Liu teach that the conjugate reagent comprises a detection binding partner for the analyte and a first small molecule (i.e., the antibody to the analyte and biotin). The conjugate reagent forms a complex with streptavidin linkers and biotinylated liposomes encapsulating labeling agents via biotin-streptavidin bond.
On the other hand, Peretz also teaches that the conjugate reagent, e.g., a binding agent of the analyte, comprises multiple biotin molecules and forms a complex with multiple streptavidin molecules (see Fig.1A). This complex is ready to bind to multiple labeling agents via a biotin-streptavidin bond, e.g., biotinylated PE. This teaching also means that the conjugate reagent of Peretz can bind to biotinylated labeling agent-encapsulated liposomes, or biotinylated labeled agents, e.g., biotinylated Pes, or the mixture thereof because the biotinylated reagents can connect to each other by the streptavidin linkers.
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 method or kit of Peretz and Liu, by further adding a labeling agent to the conjugate reagent simultaneously with the biotinylated liposome as an obvious matter to try, namely choosing from a finite list of suitable, art known method for improving analyte detection assay using streptavidin-biotin amplifying system as taught by Peretz. By adding multiple labeling agents to the conjugate reagent, a higher amplification signal will be achieved, thereby increasing the detection range of the assay (see Peretz par.5).
One having an ordinary skill in the art would have had a reasonable expectation of success in modifying because the addition of a labeling agent to the conjugate reagent is also based on the biotin-avidin bond.
Claims 12 and 14-18 are rejected under 35 U.S.C. 103 as being unpatentable over and Peretz in view of Liu, as applied in claim 11 above, and further in view of Peretz et al. (US10527614, Peretz’614).
Regarding claim 12, Peretz and Liu teach the method or kit of detecting an analyte in the sample as in claim 11. Peretz does not teach that the linker reagent comprises a labeling agent.
Peretz’614 is the patented version of the patent application of Peretz. They are directed to the same method of detecting an analyte in the sample using a streptavidin-biotin amplifying system as discussed in Peretz above (see Peretz’614 col.2 par.2). Peretz’614 teaches that, in the biotin-streptavidin amplifying system, the labeling agents can be coupled with biotin and streptavidin to enhance the signal of the analyte detection in the sample as shown in Fig.1A where one analyte can be detected by a number of labeling agents connected to each other by biotin-streptavidin bonds (see Peretz’614 col.2 lines 44-48 and Fig.1A).
Fig.1A excerpted from Peretz’614
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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 method or kit of Peretz and Liu, by further adding a labeling agent to the streptavidin linkers as taught by Peretz’614 as an obvious matter to try, namely choosing from a finite list of suitable, art known method for improving analyte detection assay using streptavidin-biotin amplifying system. By doing this, a double amplification signal will be achieved as both streptavidin and subsequent biotin are labeled, thereby increasing the detection range of the assay (see Peretz’614 col.2 par.1 and example 4).
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Peretz and Peretz’614 because they are directed to the immunoassay where the labeling agents and the analyte detector are conjugated by using biotin-avidin bond.
Regarding claim 15, Peretz, Liu, and Peretz’614 teach the method or kit of detecting an analyte in the sample as in claim 12. Peretz teaches that the sample and reagents are combined sequentially (see Peretz par.6).
Claims 14 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Peretz in view of Liu, and Peretz’614, as applied in claim 12 above, and further in view of Law (US5449556).
Regarding claim 14, Peretz, Liu, and Peretz’614 teach the method or kit of detecting an analyte in the sample as in claim 12. Peretz and Liu does not specifically teach the signaling molecule in the liposomes.
Law discloses a method for the detection of an analyte in a fluid sample using liposomes encapsulating acridinium esters (AE) (see Abstract). Law teaches that the novel acridinium esters of the present invention are highly soluble in water and can be encapsulated in liposomes at high concentrations. Once inside liposomes, the novel acridinium esters remain encapsulated for extended periods of time. See column 5 paragraph 1. Moreover, the AE encapsulated liposomes can be coupled to molecules with biological activity and used in luminescent assays (see Abstract, col.5 lines 62-68, col.6 lines 1-5: e.g., avidin).
Therefore, 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 method or kit of Peretz and Liu, substituting acridinium esters as taught by Law for the signaling molecules in the biotinylated liposome in the method or kit of Peretz and Liu because Liu is generic to a signaling molecule and Law teaches a specific signaling molecule that can be encapsulated in the liposome at high concentrations and remain encapsulated for extended periods of time.
A person of ordinary skill in the art would have been motivated to use acridinium esters in liposome because the signal to noise ratio at the low end of the assay is significantly increased, allowing better assay sensitivity, precision and speed (see Law col.13 part D).
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Peretz in view of Liu and Law because they are directed to a detection method using signaling molecule encapsulated in a liposome, wherein the AE encapsulated liposomes can be coupled to molecules with biological activity and used in immunoassays (see Abstract, col.5 lines 62-68, col.6 lines 1-5: e.g., avidin).
Regarding claim 17, Peretz, Liu, Peretz’614, and Law teach the method or kit of detecting an analyte in the sample as in claim 14. Peretz does not teach the concentration of the encapsulated acridinium ester (AE).
Law teaches that a concentration of AE (from Examples 1 to 7) used for making encapsulated AE lumisomes ranges 0.1-0.26 mg/ml (see Law Example 8). The lowest concentration AE (e.g., DMAE-ANDS) used in Law is equivalent to 12-10-6 mol/L based on the molecular weight of DMAE-ANDS 772g/mole.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to prepare the encapsulated AE at a concentration more than 1x10-6 mol/L as suggested by Law in the modified method or kit of Peretz, Liu, Peretz’614, and Law with a reasonable expectation of success because Law teaches that the hydrophilic acridinium esters are useful for encapsulation within liposomes for use as chemiluminescent markers with minimum leakage through the liposome wall at the concentration 0.1-0.26 mg/ml (see Law col.1 lines 40-60, and Examples 1-8).
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Peretz in view of Liu and Law because they are directed to a detection method using signaling molecule encapsulated in a liposome, wherein the AE encapsulated liposomes can be coupled to molecules with biological activity and used in immunoassays (see Abstract, col.5 lines 62-68, col.6 lines 1-5: e.g., avidin).
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Peretz in view of Liu, and Peretz et al. (US10527614, Peretz’614), as applied in claim 12 above, and further in view of Hirt et al. (US20090325171).
Regarding claim 16, Peretz, Liu, and Peretz’614 teach the method or kit of detecting an analyte in the sample as in claim 12. Peretz does not teach the size of the liposome.
Liu teaches that the liposomes has nano size (see Title and Abstract teaching nano-biotinylated liposome, page 472 col.2 par.2 teaching the biotinylated liposome has a hollow closed-shell nanosphere).
Hirt teaches a method of detecting an analyte in a sample using fluorophore encapsulated liposomes, wherein such liposomes can be covalently linked to antibodies and their fluorescence measured upon binding to the antigen (see par.18). Hirt also teaches that liposomes with diameters of 100 nm can contain up to 25,000 fluorophore, so the analyte detection can be achieved at nanomolar sensitivity (see par.18).
Liu is generic for the size of the liposomes at nanoscales and Hirt provides a specific size of liposome at 100 nanometers. The liposomes taught by Liu and Hirt are used as signal amplifying systems in an immunoassay. While Liu and Hirt do not specifically teach the claimed diameter of the liposomes, the liposome amplifying system taught by the prior arts can be used to improve the sensitivity of immunoassay (see Hirt par.18, Liu page 472 col.2 par.1 teaching that liposome-encapsulated signals can produce 500–1000-fold signal enhancement).
Absent unexpected results, it would have been obvious to one of ordinary skill to have arrived at the claimed diameter of the liposomes by routine optimization in order to uncover the optimum workable ranges of diameter of the liposomes used in the immunoassay. One having ordinary skill in the art would have a reasonable expectation of success in arriving at the claimed range through routine optimization because the prior art specifically discloses that Nano-biotinylated liposome-based immunoassay can be used for the ultrasensitive detection of protein marker in a sample (see Liu Title).
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Peretz in view of Liu, Peretz’614, and Law, as applied in claim 14 above, and further in view of Hirt et al. (US20090325171).
Regarding claim 18, Peretz, Liu, Peretz’614, and Law teach the method or kit of detecting an analyte in the sample as in claim 14. Peretz does not teach a liposome can contain up to 1000-100,000,000,000 AE molecules.
Hirt teaches a method of detecting an analyte in a sample using fluorophore encapsulated liposomes, wherein such liposomes can be covalently linked to antibodies and their fluorescence measured upon binding to the antigen (see par.18). Hirt also teaches that liposomes with diameters of 100 nm can contain up to 25,000 fluorophore, so the analyte detection can be achieved at nanomolar sensitivity (see par.18).
Therefore, it would have been obvious to one having an ordinary skill in the art before the effective filing date of the claimed invention to modify the method or kit of Peretz, Liu, Peretz’614, and Law, by encapsulating about 25,000 AE molecules in the liposome as taught by Hirt, as an obvious matter to try, namely choosing from a finite list of suitable, art recognized/known liposome-based amplifying reagent for improving the sensitivity of the immunoassay (see Hirt par.18, Liu page 472 col.2 par.1 teaching that liposome-encapsulated signals can produce 500–1000-fold signal enhancement, Law Example 13 parts D-E).
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Peretz in view of Liu, Law and Hirt because they are directed to a detection method using signaling molecules encapsulated in a liposome as a amplifying reagent.
Response to Arguments
Applicant’s arguments with respect to claim 2 have been considered but are moot because the new ground of rejection is made in view of the amendment of the claim.
Applicant’s arguments with respect to claims 1,3-5 have been considered but are moot because the new ground of rejection is made in view of the amendment of the claim.
Conclusion
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CHAU N.B. TRAN whose telephone number is (571)272-3663. The examiner can normally be reached Mon-Fri 8:30-6:30 CT.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bao-Thuy L Nguyen can be reached at 571-272-0824. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/CHAU N.B. TRAN/Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/Supervisory Patent Examiner, Art Unit 1677 May 20, 2026
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