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 .
DETAILED ACTION
Action Is Final, Necessitated by Amendment
Applicants' response to the Non-Final Office Action mailed 15 April 2025, has been entered and the Remarks therein, filed 15 July 2025, are fully considered here.
This action is a Final Office Action, based on new grounds under 35 U.S.C. §112(b) and under 35 U.S.C. §103 over Jin in view of Hermetter, Oakeshott et al., and Reineke et al., necessitated by Applicants’ amendment received 15 July 2025, specifically, amended claim 1. See MPEP 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
Status of Claims
Claims 1-5, 7-14 and 18-22 are pending.
Claims 9-14 and 18-22 are withdrawn from consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected groups, according to a previous Primary Examiner. Election is considered to have been made without traverse during the telephonic correspondence that the previous Primary Examiner held on 13 April 2023 with Applicant.
Claims 1-5, 7 and 8 are rejected.
Claim 1 is objected to.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. §119(e) or under 35 U.S.C. §120, §121, or §365(c) is acknowledged. This application claims benefit of 62/955,156, filed 12/30/2019.
Applicant has complied with all of the conditions for receiving the benefit of an earlier filing date under 35 U.S.C. §120 or §365(c).
Claims 1-5, 7 and 8 have the effective filing date of 30 December 2019.
Claim Objections
Claim 1 is objected to because of the following informalities:
Claim 1 recites: "...; and wherein the kit does not contain Tris(hydroxymethyl)aminomethane hydrochloride to ensure stable pH regardless of temperature", which should read: "...; and wherein the kit does not contain Tris(hydroxymethyl)aminomethane hydrochloride to ensure a stable pH regardless of temperature."
Appropriate correction is required.
Claim Rejections - 35 U.S.C. § 112
35 U.S.C. § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. §112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. §112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
35 U.S.C. §112(a) and pre-AIA 35 U.S.C. §112, first paragraph require that the specification include the following (MPEP 2161 (I)):
(A) A written description of the invention;
(B) The manner and process of making and using the invention (the enablement requirement); and
(C) The best mode contemplated by the inventor of carrying out his invention.
Claims 1-5, 7 and 8 are rejected under 35 U.S.C. §112(a) or 35 U.S.C. §112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contain(s) subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
[Claims 2-5, 7 and 8 are dependent on claim 1, contain the limitations of claim 1, and, therefore, are rejected for the same reason.]
Claims 1-5, 7 and 8 fail to comply with the written description requirement because they contain new matter.
Claims 1-5, 7 and 8 fail to comply with the written description requirement, because the claim text(s) recite(s) limitations which are not described in the specification and/or which encompass a claim breadth which is not supported by the specification.
Claim 1 recites: "...; and wherein the kit does not contain Tris(hydroxymethyl)aminomethane hydrochloride to ensure stable pH regardless of temperature."
The specification recites: "The kits of the invention do not contain sodium chloride or Tris(hydroxymethyl)aminomethane hydrochloride" (originally-filed specification, pg. 12, para. [0044]). That is, the specification explains that the kit(s) of the claimed invention do not contain Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), but it does not explain that the exclusion of Tris-HCl from the kit(s) ensures a stable pH regardless of temperature.
In the Declaration filed 24 September 2024, one of the inventors Yanping Chen explains that: "Tris-HCl is avoided in our honey bee-specific buffer due to its temperature dependent pH variability. Tris (Hydroxymethyl) Aminomethane Hydrochloride (Tris-HCl) is temperature-sensitive, meaning the pH of a Tris buffer changes with fluctuations in temperature. This is especially important in assays involving honey bees, where the temperature of the assay may need to reflect physiological conditions (e.g., ~30-35°C)...Alternative buffers like PBS, which maintains better stability at physiological pH, are preferred to avoid the limitations of Tris" (Dec., pg. 3, para. 8). However, this does not explain how a kit not containing Tris-HCl ensures a stable pH; on the other hand, it is the presence of PBS (phosphate buffered saline) that ensures a stable pH (over a limited temperature range). It is noted that the instantly-claimed kit consists of components or reagents which only contain PBS.
It is not clear that Applicant has support for 'ensuring a stable pH regardless of temperature'. That is, Applicant does not have support for (nor is Applicant enabled for) a kit which contains components which ensure a stable pH over any and all temperatures. As noted above, Declarant states that the assay (which is performed using the claimed kit) may potentially be conducted over a temperature range of ~30 to 35oC. The specification describes that lipase activity was measured at 37oC (spec., pg. 23, para. [0083], Example 1).
Reineke et al. ((2011) Intl. J. Food Prop. 14: 870-881) (cited in the 103 rejection below) measures the pH shift among TRIS, ACES and PBS buffer solutions at a temperature range of 20-130oC (pg. 870, Abstract). Reineke et al. further teaches that extensive data about the pH value of various kinds of foods at ambient temperature are available, and some data covers pH values for buffer solutions up to 80◦C, or even 90◦C for some foods; however, there are limited data available about the shift of the pH values up to 130◦C (pg. 871, para. 2). That is, it is not known in the art that any buffer (including PBS) can ensure a stable pH at any temperature, particularly temperatures over the 130oC (or under the 20oC) tested by Reineke et al.
To overcome this rejection, Applicant may attempt to demonstrate (by means of argument or evidence) that the original disclosure establishes that he or she was in possession of the amended claim, the claim may be amended to recite a property or characteristic of the kit that is supported by the specification or the amendment reciting the 'wherein'-type clause may be deleted.
35 U.S.C. § 112(b)
The rejection of Claim 8 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 pre-AIA the applicant regards as the invention, in the Non-Final Office Action mailed 15 April 2025, is withdrawn in view of Applicants’ amendment received 15 July 2025, in which the cited claim was amended.
The following is a quotation of 35 U.S.C. §112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. §112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-5, 7 and 8 are rejected under 35 U.S.C. §112(b) or 35 U.S.C. §112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention.
[Claims 2-5, 7 and 8 are dependent on claim 1, contain the limitations of claim 1, and, therefore, are rejected for the same reason.]
[This rejection cited in view of Applicant's amendment.]
Claims 1-5, 7 and 8 are indefinite because the metes and bounds of the claimed subject matter are not clear.
Claim 1 recites the relative term "consistently".
Claim 1 recites: "...; wherein the lipase activity quantified in the honey bee biological sample using the kit consistently correlates with the honey bee stress;..."
However, it is not clear to what extent (or how often) quantified lipase activity correlates with honey bee stress, within the context of the claimed subject matter or for the purpose of finding relevant prior art.
The term 'consistently' is a relative term which renders the claim indefinite. The term ‘consistently’ is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. There is no definition, explanation or description given for the term 'consistently' (MPEP 2173.05 (b)(I)). This term will be defined by its plain meaning at the discretion of the Examiner, as it applies to the claim language (see MPEP 2111.01).
An American English dictionary definition of the word 'consistently' is: with no or very few exceptions; typically; usually.
Therefore, the interpretation of the claimed subject matter is that the quantified lipase activity in the majority of cases (but, in some cases, may not) correlate(s) with honey bee stress.
Claim Interpretations
(1) Claim 1 contains intended use language and inherent functional claim language.
Claim 1 recites: “A kit for determining honey bee stress by quantifying lipase activity in a honey bee biological sample, the kit consisting of:…; wherein the lipase activity quantified in the honey bee biological sample using the kit consistently correlates with the honey bee stress; and wherein the kit does not contain Tris(hydroxymethyl)aminomethane hydrochloride to ensure stable pH regardless of temperature.”
Claim 1 recites an intended use in the claim preamble.
The phrase as an intended use merely states the purpose or intended use of the invention, rather than any distinct definition of any of the claimed invention's limitations. Also, the intended use terminology does not limit the structure or method steps of the claimed invention. Therefore, the phrase as an intended use does not limit the scope of the claimed subject matter. (See MPEP 2111.02 (I)(II).) That is, the intended use recited in the preamble does not result in a structural or manipulative difference between the claimed invention and the prior art. Deletion of the preamble phrases does not affect the structure or steps of the claimed invention (MPEP 2111.02 (II)).
With regard to the ‘wherein-type’ clauses, it is well known that claim scope is not limited by claim language that suggests or makes optional, but does not limit a claim to a particular structure. In addition, when a structure recited in the reference is substantially identical to that of the claims, claimed properties or functions are presumed to be inherent (MPEP 2112.01 (I)). In addition, it is well known that “[T]he discovery of a previously unappreciated property of a prior art composition, or of a scientific explanation for the prior art’s functioning, does not render the old composition patentably new to the discoverer.” Atlas Powder Co. v. Ireco Inc., 190 F.3d 1342, 1347, 51 USPQ2d 1943, 1947 (Fed. Cir. 1999). Thus the claiming of a new use, new function or unknown property which is inherently present in the prior art does not necessarily make the claim patentable. In re Best, 562 F.2d 1252, 1254, 195 USPQ 430, 433 (CCPA 1977) (MPEP 2112 (I)).
For the purpose of compact prosecution, the claim will be interpreted to mean that any combination of the three instantly-claimed kit components cited in the prior art will be considered to be able to quantify honey bee lipase activity which will be considered to (inherently) correlate with the level of honey bee stress. In addition, any combination of the three instantly-claimed kit components cited in the prior art (i.e., which does not include Tris-HCl) will be considered to (inherently) ensure stable pH regardless of temperature.
Prior art will be applied according to this interpretation.
Prior art which shows that lipase activity can be quantified in a honey bee biological sample will be indicated at the discretion of the Examiner.
(2) Claims 1, 5 and 7 cite the term “n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate”.
It is noted that previously-filed claims had originally cited the term ‘Zwittergent 3-16’ (in lieu of the above cited term) which was deemed to be a registered trademark name. Therefore, Applicant replaced the term ‘Zwittergent’/’Zwittergent 3-16’ for its analogous chemical name ‘n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate’.
For example, Jin et al. (cited in the Final Office Action mailed 27 November 2024 and in the 103 rejection below) shows: “The second buffer can comprise n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate or Zwittergent®” (pg. 2, para. [0023]).
Therefore, prior art which shows the term ‘Zwittergent’ or any variation thereof, as cited within the context of the claimed subject matter, will be considered to be applicable prior art with regard to the chemical name now recited in the instant claims.
(3) Claim 4 cites the term “4,4 difluoro-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid dye”.
It is noted that previously-filed claims had originally cited the term ‘BODIPY’ (in lieu of the abovecited term) which was deemed to be a registered trademark name. Therefore, Applicant replaced the term ‘BODIPY’ for its analogous chemical name ‘4,4 difluoro-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid’.
For example, Jin et al. (cited in the Final Office Action mailed 27 November 2024 and in the 103 rejection below) shows: “The substrate can comprise an EnzChek® lipase substrate which is composed of a 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY®) dye…” (pg. 2, para. [0024]).
Therefore, prior art which shows the term ‘BODIPY’ or any variation thereof, as cited within the context of the claimed subject matter, will be considered to be applicable prior art with regard to the chemical name now recited in the instant claims.
(4) Claims 3 and 4 cite the term “4,4 dimethylamino-phenyl-azo-benzoic acid”.
Claim 3 also describes the analogous term ‘DABCYL acid’ which was deemed to be a registered trademark name. Therefore, Applicant had replaced the term ‘DABCYL acid’ for its analogous chemical name ‘4,4 dimethylamino-phenyl-azo-benzoic acid’ in claim 4.
Therefore, prior art which shows the term ‘DABCYL’ or any variation thereof, as cited within the context of the claimed subject matter, will be considered to be applicable prior art with regard to the chemical name now recited in the instant claims.
Claim Rejections - 35 U.S.C. § 103
The rejection of Claims 1-5, 7, 8 and 23 under 35 U.S.C. §103 as being unpatentable over Jin in view of Hermetter, and Oakeshott et al., in the Non-Final Office Action mailed 15 April 2025, is withdrawn in view of Applicants' amendment received 15 July 2025.
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 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 of this title, 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.
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.
Claims 1-5, 7 and 8 are rejected under 35 U.S.C. §103 as being unpatentable over Jin (U.S. Patent Application Publication No. 2012/0064556 A1) in view of Hermetter (Triglyceride Lipase Assays. In: Lipase and Phospholipase Protocols. Copyright 1999; pp. 19-29), Oakeshott et al. (U.S. Patent Application Publication No. 2005/0176118 A1), and Reineke et al. ((2011) Intl. J. Food Prop. 14: 870-881).
[All references except Reineke et al. cited in the Non-Final Office Action mailed 15 April 2025.]
[This rejection cited in view of Applicant's amendment.]
Jin addresses some of the limitations of claim 1.
Regarding claim 1, pertaining to a kit for determining honey bee stress by quantifying lipase activity in a honey bee biological sample consisting of buffers containing bovine serum albumin (BSA) and/or an n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, wherein the lipase activity quantified in the honey bee biological sample using the kit consistently correlates with the honey bee stress,
[See Claim Interpretations section- (1) and (2) above.]
Jin shows a method and kit for the measurement of LPL (lipoprotein lipase) (pg. 1, para. [0010]). Once a biological sample has been gathered from a subject it is added to a well of a well plate. After the biological sample has been added to the well of the well plate, a first buffer is added. The first buffer can comprise NaCl, Tris-HCI (pH=8.0) and fatty acid free BSA (bovine serum albumin). After the first buffer is added to the well of the well plate, a second buffer is added to the same well. The second buffer can comprise n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate or Zwittergent®. The concentration of Zwittergent® can be at any suitable level, such as about 0.0125% (pg. 2, para. [0022] thru [0023]).
Further regarding claim 1, pertaining to the kit consisting of a dye and a quencher in a triglyceride backbone,
Jin shows that following the addition of the second buffer, a substrate is added to the same well. The substrate can comprise an EnzChek® lipase substrate which is composed of a 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY®) dye and a quencher in a TG (triglyceride) backbone (pg. 2, para. [0024]).
Jin does not show: 1) BSA and/or n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate in phosphate buffered saline (PBS), including 4X PBS [Claim 1].
Hermetter addresses some of the limitations of claim 1.
Hermetter shows triglyceride lipase assays based on a novel fluorogenic substrate (pg. 19, Title [nexus to Jin- triglyceride lipase assays using fluorogenic substrates]).
Regarding claim 1, pertaining to phosphate buffered saline (PBS),
Hermetter shows that recommended buffers and/or buffer reagents for determining the enzymatic activity of lipases are listed and include, minimally, Tris-HCI or phosphate-buffered saline (PBS) (pg. 24, para. 2.3, entry# 3).
Oakeshott et al. provides information that would have motivated one of ordinary skill in the art to have used the lipase assay, shown by Jin et al. in view of Hermetter, to quantify lipase in a honey bee biological sample, by way of addressing the limitations of claim 1.
Oakeshott et al. shows the use of lipases and esterases as catalysts in biotransformation processes (pg. 1, para. [0001] [nexus to Jin- lipases]).
Regarding claim 1, Oakeshott et al. shows that the described invention provides an enzyme-based biocatalysis process, wherein the enzyme is an insect esterase or lipase (pg. 3, para. [0024]). Oakeshott et al. also shows the use of a fluorogenic assay to measure the lipase activity of insect esterases or lipases (pg. 17, para. [0219] thru [0220] [nexus to Jin- lipase assay using a fluorescent dye]).
Oakeshott et al. further shows, with regard to the use of PBS as a lipase assay reagent [Claim 1], that compositions useful for the processes of the described invention which comprise an insect lipase, include excipients such as water, saline, and Ringer’s solution and other aqueous physiologically balanced salt solutions. Excipients can also contain additives, such as substances that enhance isotonicity and chemical stability, which include phosphate buffer or Tris buffer (pg. 10, para. [0145]).
Reineke et al. provides information that would have motivated one of ordinary skill in the art to have not supplied Tris(hydroxymethyl)aminomethane hydrochloride in a kit containing reagents for a lipase assay, such as the assay shown by Jin et al. in view of Hermetter, and Oakeshott et al., in order to ensure stable pH regardless of temperature, by way of addressing the limitations of claim 1.
It is noted that the references of Hermetter, and Oakeshott et al. show that alternative buffers to Tris-HCl/Tris, including PBS, can be used in the lipase assay. Therefore, the references inherently show a list of lipase assay ingredients or reagents which do not contain Tris-HCl/Tris.
Regarding claim 1, Reineke et al. teaches that when the temperature increases after an initial measurement of the pH at ambient temperature (25◦C), a significant pH shift could occur, which could produce incomparable results in different buffer solutions. Reineke et al. shows a study in which a measurement cell was constructed to record online the pH-value and temperature up to 130◦C. The pH shift was measured over a wide temperature range (ΔT 20–130◦C) in the most commonly used buffer solutions. The Δ pH of certain buffer solutions, namely TRIS and ACES, showed a significant pH decrease of −2.01 ± 0.08 (ΔT 20–130◦C) and −1.27 ± 0.1 (ΔT 20–130◦C), respectively, whereas the pH of PBS buffer solution was nearly independent of temperature (pg. 870, Abstract).
Reineke et al. further teaches that if this pH change is not considered during the experimental design, it could produce a much stronger inactivation of the microorganism or enzyme incorporated in the TRIS or ACES buffer solution (pg. 876, lines 8-10). With the pH shifts measured in different buffer solutions, the results of microbial and enzymatic inactivation experiments can be compared more effectively and the influence of the temperature-dependent pH shift could be controlled by changing the buffer solution (pg. 879, para. 2).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have modified the kit consisting of a dye and a quencher in a triglyceride backbone, a substrate reaction buffer comprising n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and a buffer comprising n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and bovine serum albumin (BSA), as shown by Jin, by substituting the Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) and sodium chloride (NaCl) shown by Jin, with the phosphate buffered saline (PBS) buffer [Claim 1], shown by Hermetter, with a reasonable expectation of success, because Hermetter teaches that a lipase assay buffer can include Tris-HCl or PBS (MPEP 2143 (I)(B)(3)). In addition, the lipase assay shown by Hermetter is a fluorogenic assay, which is the same type of lipase assay as shown by Jin (MPEP 2143 (I)(G)).
In addition, with regard to the limitation that the kit does not contain Tris(hydroxymethyl)aminomethane hydrochloride to ensure stable pH regardless of temperature [Claim 1], because the prior art references of Hermetter, and Oakeshott et al. show alternative buffer selections (e.g., Tris or PBS), it would have been obvious to have not included Tris buffer in the kit (MPEP 2143 (I)(G)).
On the other hand, Reineke et al. shows that PBS is a more stable buffer than Tris, with regard to buffering capacity during temperature fluctuations over time. Therefore, Reineke et al. provides motivation for substituting the Tris-HCl, shown by Jin, with the PBS, shown by Hermetter (and Oakeshott et al.), with a reasonable expectation of success (MPEP 2143 (I)(G)).
However, even in the absence of Reineke et al., it would have been obvious to have not included Tris in the claimed kit (for any reason), because the prior art references of Hermetter, and Oakeshott et al. show alternative buffer selections (e.g., Tris or PBS) which can be used in the lipase assay, as noted above.
It would have been further obvious to have used the kit shown by Jin to have assayed for insect lipase activity [Claim 1], as shown by Oakeshott et al., with a reasonable expectation of success, because Oakeshott et al. shows that insect lipases can be assayed for activity using a fluorogenic substrate, which is the fluorogenic lipase assay shown by Jin (MPEP 2143 (I)(G)).
Even in the absence of Oakeshott et at., it would have been obvious to one of ordinary skill in the art of performing lipase assays to have used the reagents shown by Jin and Hermetter (and as instantly-claimed) to determine lipase activity in an insect biological sample [Claim 1], with a reasonable expectation of success, barring a showing that the kit reagents, as instantly-claimed, would not be expected to successfully assay lipases from other biological sources (MPEP 2144 (III)).
One of ordinary skill in the art would have been motivated to have made those modifications, because Jin teaches that the EnzChek® substrate can be used for the accurate and sensitive detection of LPL activity in solution. Thus, the BODIPY® dye can be used for accurate and sensitive detection of LPL activity in solution without the need of a purification step (Jin, pg. 2, para. [0026]). In addition, the BODIPY® (fluorescence) spectrum is relatively insensitive to solvent polarity and is stable in the physiological pH range, which is an advantage over 7-nitrobenz-2-oxa-1,3-diazole (NBD) labeled substrates. BODIPY® has a sufficient photostability and can be handled without need of being shielded from the light. BODIPY® also does not have an ionic charge and will not interfere with the solubility of TG into which it is incorporated (Jin, pg. 2, para. [0024]).
Compare to Applicant’s use of the EnzChek® lipase substrate (as also shown by Jin et al.) which is composed of a BODIPY dye and a DABCYL acid quencher in a triglyceride backbone (originally-filed specification, pg. 22, para. [0078]).
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention.
Jin further addresses some of the limitations of claims 5, 7 and 8, and the limitations of claims 2, 3 and 4.
Regarding claim 3, pertaining to DABCYL acid, and
regarding claims 2 and 4,
[See Claim Interpretations section- (3) and (4) above.]
Jin shows that the substrate can comprise an EnzChek® lipase substrate which is composed of a 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY®) dye and a quencher in a TG (triglyceride) backbone. The substrate, which includes the fluorescent dye BODIPY®, can be commercially purchased from Invitrogen™. In the presence of a quencher, the substrate is a stable and nonfluorescent compound in an un-reacted state, but produces a green fluorescent BODIPY® labeled fatty acid in the presence of LPL. BODIPY® labeled substrate does not exhibit fluorescence in its unhydrolyzed state due to a bridged quencher (Dabcyl) on the adjacent fatty acid arm (pg. 2, para. [0024] thru [0025]).
Regarding claims 5 and 7, pertaining to the concentrations of the BSA and/or the n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,
[See Claim Interpretations section- (2) above.]
Jin shows that the concentration of BSA can be at any suitable level, such as about 6% BSA. The concentration of Zwittergent® can be at any suitable level, such as about 0.0125% (pg. 2, para. [0022] thru [0023]).
Regarding claim 8, Jin shows that the kit would include a first buffer. The first buffer could comprise a mixture of NaCl, Tris-HCl and fatty acid free BSA. The kit would further include a second buffer. The second buffer could comprise n-Hexadecyl-N,N-dimethyl- 3-ammonio-1-propanesulfonate or Zwittergent®. Further included in the kit is a substrate. The substrate can be EnzChek® lipase substrate which is composed of a 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY) dye and a quencher in a TG backbone (pg. 3, para. [0037] thru [0038]).
It would have been further obvious to have used routine optimization in order to have determined the optimal amounts of n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (e.g., 0.005% or 0.06%) and bovine serum albumin (BSA) (e.g., 0.0015%) to put in the kit [Claims 5 and 7], barring a showing of criticality for the specific limitations, because Jin shows specific concentrations of both Zwittergent (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) (i.e., about 0.0125%) and BSA (i.e., about 6%) and teaches that the concentrations can be at any suitable level (pg. 2, para. [0023]) (MPEP 2144.05 (II)(A) and (III)(A)). In addition, Jin shows that the optimal concentration of reagents was determined by performing a titration experiment with different amounts of LPL (pg. 2, para. [0028]).
It would have been further obvious to have provided the various kit reagents (i.e., the sample buffer, the substrate reaction buffer, and the fluorogenic triglyceride comprising a dye and a quencher) in separate containers [Claim 8], because, although not explicitly stated in Jin, the reference recites the piecemeal addition of the various reagents or ingredients into the kit. One of ordinary skill in the art of kit preparation would have provided the reagents (e.g., for an assay) in separate containers so that the enduser could dilute each one to a specific concentration depending on the particulars of the assay to be conducted (e.g., the amount of enzyme to be detected, in the case of a lipase assay or the amount of honey bee biological sample as substrate). In addition, it would be obvious to provide the various buffers/reagents in separate containers to prevent a premature chemical reaction between the various reagents prior to the addition of the lipase.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention.
Claim 1 is rejected under 35 U.S.C. §103 as being unpatentable over Haller et al. ((2017) J. Lipid Res. 58: 1166-1173) as evidenced by Basu et al. ((2011) J. Lipid Res. 52: 826-832).
[This rejection cited in the Non-Final Office Action mailed 15 April 2025.]
Haller et al. as evidenced by Basu et al. addresses some of the limitations of claim 1.
Regarding claim 1, Haller et al. shows the measurement of LPL (lipoprotein lipase) using the EnzChek fluorogenic substrate. A 2× reaction mix was prepared freshly for each assay containing 4 mg/ml BSA, 0.025% Zwittergent 3-14, 7µM EnzChek substrate, 200 nM APOC2 in phosphate saline buffer pH 7.5 with 1 mM CaCl2 and 0.5 mM MgCl2. The solution was mixed 1:1 with samples, fluorescence was measured every 30 s (pg. 1167, column 2, para. 4).
Haller et al. does not specifically show that the EnzChek fluorogenic substrate contains a dye and a quencher in a triglyceride backbone, with regard to claim 1.
Basu et al. shows a novel, real-time, homogeneous fluorogenic lipoprotein lipase (LPL) assay using the commercially available substrate, the EnzChek lipase substrate, which is solubilized in Zwittergent (pg. 826, column 1, Abstract [nexus to Haller et al.- EnzChek lipase assay]). A fluorescent triglyceride (TG) derivative was sought as a substrate because a TG-like molecule should be stable and soluble in the conditions under which LPL functions. The EnzChek lipase substrate was a very attractive possibility, containing the fluorescent BODIPY-C12 FA derivative in ester linkage at the sn -1 position of glycerol, a BODIPY fluorescent quencher FA derivative, Dabcyl, at the sn -2 position, and a C-6 FA aliphatic chain in ether linkage at sn -3 (pg. 827, column 2, para. 3 [dye = BODIPY; quencher = Dabcyl]).
Haller et al. as evidenced by Basu et al. does not show: 1) a kit comprising the components shown by Haller et al. (i.e., not Tris/HCl) [Claim 1]; and 2) an n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate in Phosphate Buffered Saline (PBS) substrate reaction buffer, including a 4X PBS buffer [Claim 1]
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have combined the list of components for determining LPL activity, shown by Haller et al. and Basu et al., into a kit [Claim 1], with a reasonable expectation of success, because one of ordinary skill in the art would have understood that any combination of components or reagents employed in an assay or other laboratory protocol could be combined into a kit format for the reason that kits are well known in the laboratory protocol arts for providing convenience to the enduser, thereby expediting the performance of any laboratory protocol (MPEP 2144 (I)).
It would have been further obvious to have formulated the Zwittergent reagent, shown by Haller et al. and Basu et al., in a PBS buffer [Claim 1], with a reasonable expectation of success, because Haller et al. shows that other reagents (e.g., APOC2) in the assay are diluted in phosphate buffered saline, and yet the protocol could be performed successfully (MPEP 2143 (I)(G)). Therefore, it would have been obvious to have prepared the Zwittergent reagent in BSA, as shown by Haller et al., as well as PBS.
It would have been further obvious to have formulated the Zwittergent reagent in a 4x PBS buffer [Claim 1], because one of ordinary skill in the art of conducting laboratory assays would have used routine optimization in order to have determined the optimal strength of the PBS buffer (e.g., as a 4x buffer) suitable for the particulars of the specific assay (e.g., depending on the amount or type of sample to be assayed or type of fluorogenic substrate used), barring a showing of criticality for the specific limitation(MPEP 2144.05 (II)(III)).
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention.
Response to Arguments
Applicant’s arguments, pp. 7-12, filed 15 July 2025, with respect to the claim interpretations and the prior art references cited in the 35 U.S.C. §103 rejections, have been fully considered but they are either not persuasive or are moot because the arguments do not apply to the references as they are applied in the context of the current rejection, or as new grounds necessitated by Applicant’s amendment, in which claim 1 was amended.
1. Applicant remarks (pg. 7, last para.), with regard to the claim interpretations, that Applicant submits that the 132 Declaration by Dr. Chen filed on 24 September 2024 (hereinafter "the Chen Declaration"), teaches that only when using the claimed kit with the specific chemicals was it possible to consistently measure honeybee lipase consistently and for the lipase activity measurements to reflect the honeybee stress levels. One of the basic and novel characteristics of the presently claimed kit and method is that only when the kit consists of the claimed active ingredients, is it possible to consistently measure honeybee lipase activity, and correlate the measured lipase activity value with honeybee stress. Thus, any prior art that recites any of the terms "Zwittergent," "BODIPY," "DABCYL," or a variation thereof and recites at least one additional active ingredient is not applicable prior art.
However, in response to Applicant, earlier-filed claims (e.g., submitted 28 December 2020) recited the terms 'Zwittergent', 'BODIPY', and 'DABCYL'. Although these terms have now been described by their chemical names (and not by their tradenames or trademarks), the chemicals or reagents described by these tradenames or trademarks are applicable prior art. In addition, the Chen Declaration (as previously-responded to) appeared to emphasize the preferential use of phosphate-buffered saline (PBS) instead of Tris in the lipase assay. However, it is well known in the art (see the newly-cited reference of Reineke et al. in the 103 rejection above) that there are advantages to using PBS vs Tris in some assays; this will be discussed in further detail below.
2. Applicant remarks (pg. 9, para. 6 thru pg. 10, para. 4), with regard to the 103 rejection(s), that, as stated above and in the Chen Declaration, only when using the claimed kit was it possible to measure honeybee lipase consistently and for the lipase activity measurements to reflect the honeybee stress levels. Jin teaches lipase quantitation using a first buffer containing NaCl, Tris, BSA, Zwittergent, a non-water soluble substrate, and a correlator. Hermetter uses assay buffers for the determination of lipase activity contain Tris or PBS at pH 7.4, KCl, KH2PO4, NaCl, Na2HPO4. Oakeshott uses the fluorescence released when 4-methylumbelliferyl palmitate is hydrolyzed in a PBS buffer containing detergent.
Applicant further remarks that the Chen Declaration teaches that honeybees have developed sophisticated thermoregulatory mechanisms to maintain a more constant body temperature. The Chen Declaration continues that Tris-HCl is temperature-sensitive, and thus, avoided in the bee specific buffers for the kit to maintain better stability at physiological pH. The Chen Declaration teaches that only a kit and method having the claimed active ingredients will consistently measure honeybee lipase activity, and this lipase activity is an indication of honeybee stress. Following the teachings of Jin and Hermetter, or of Jin and Oakeshott, a person of skill in the art will not predictably and successfully arrive at the claimed kit or the claimed kit as a whole. Prior to the instant application a person of skill in the art would not have known to avoid Tris-HCl in the compositions of a kit for consistently measuring honeybee lipase activity, and determining the level of stress of the honeybee based on this measurement.
However, in response to Applicant, Jin shows a fluorogenic lipase assay which includes all of the reagents cited in instant claim 1 (which are components of the commercially-available EnzChek® lipase assay) except for the use of phosphate-buffered saline (PBS). (It is noted that Applicant also uses the EnzChek® lipase assay to quantify lipase in a honey bee biological sample.) Hermetter et al. shows a fluorogenic lipase activity assay using PBS (or other buffers, including Tris-HCl). Oakeshott et al. also shows a fluorogenic lipase assay (including for insect lipase) which can incorporate phosphate buffer or Tris buffer, and shows working examples in which phosphate buffer is used with or without Triton X-100.
Further in response to Applicant, the potential variability in the buffering capacity of Tris under different temperatures is taught by the newly-cited reference of Reineke et al. (as noted in the 103 rejection above). That is, Reineke et al. shows that the change in pH of certain buffer solutions, including TRIS, showed a significant pH decrease (e.g., −2.01 ± 0.08 (ΔT 20–130◦C), whereas the pH of a PBS buffer solution was nearly independent of temperature (Reineke et al., pg. 870, Abstract). Reineke et al. also stresses the need for pH stability in experiments in which enzymes (or microorganisms) are used (pg. 876, lines 8-10). Therefore, one of ordinary skill in the art of assaying lipase in a honey bee biological sample, so as to quantify its activity in a repeatable manner, would have (given the choice) preferred to have used PBS, whether or not one was aware of the thermoregulatory mechanisms used by honey bees to maintain constant body temperature.
3. Applicant remarks (pg. 11, para. 3 thru pg. 12), with regard to the 103 rejection of claim 1 over Haller et al. and Basu et al., that Haller shows the measurement of lipoprotein lipase using EnzChek fluorogenic substrate: a 2x reaction mix contained 4 mg/mL BSA, 0.025% zwittergent; 7M EnzChek; 200 nM APOC2 in PBS, pH 7.5 with 1 mM CaCl2 and 0.5 mM MgCl2. Basu is said to show a lipase assay using EnzChek solubilized in Zwittergent. Both Haller and Basu measure lipase. In an effort for compact prosecution, the claims have been amended as taught above. Applicant respectfully submits that none of Haller or Basu, alone or in combination, teach or suggest the claims as a whole.
However, in response to Applicant, both Haller et al. and Basu et al. show the quantification of lipase using the commercially-available EnzChek® lipase assay. This is the same lipase assay described by Applicant (originally-filed specification, pg. 21, para. [0077], Example 1; pg. 24, para. [0085], Example 2; pg. 25, para. [0090], Example 3; pg. 26, para. [0093], Example 4; and pg. 29, para. [0104], Example 7). Haller et al. shows the use of PBS to solubilize the lipase detection reagent APOC2, which is placed in the EnzChek® substrate (Haller et al., pg. 1167, column 2, para. 4). The reagents also include BSA, Zwittergent 3-14, CaCl2 and MgCl2. Basu et al. uses the same EnzChek® lipase assay, but incorporates NaCl, Tris-HCl, EnzChek substrate, Zwittergent, and BSA (Basu et al., pg. 829, column 1, para. 1). That is, the EnzChek® lipase assay is successfully employed in both references; however, PBS is used in Haller et al. and Tris is used in Basu et al.
Therefore, it would have been obvious to one of ordinary skill in the art of quantifying lipase activity using the EnzChek® lipase assay to have used either PBS or Tris in the assay format. Therefore, a kit consisting of several of the EnzChek® assay reagents, as cited in instant claim 1, could have also included either PBS or Tris, but not necessarily both. The reference of Reineke et al. provides motivation for preferentially selecting PBS over Tris/TrisHCl as a reagent in a lipase assay kit.
Conclusion
No claims are allowed.
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 extension fee 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 date of this final action.
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/SMP/ Examiner, Art Unit 1651
/Adam Weidner/ SPE, Art Unit 1651