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 .
Status of the Claims
Claims 1, 7, 8, 11-15, 17-20 are amended. Claim 9 is cancelled.
Claims 1-8 and 10-20 are pending (claim set filed 05/27/2025). Claim 20 was withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 07/29/2024.
Claims 1-8 and 10-19 as filed 05/27/2025 are examined on the merits herein.
Withdrawal of Rejections
The response and amendment filed on 05/27/20245 are acknowledged. All of the amendment and arguments have been thoroughly reviewed and considered.
For the purposes of clarity of the record, the reasons for the Examiner's withdrawal and/or maintaining if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner's response to arguments section.
The previous claim 7 objection has been withdrawn necessitated by amendment of claim 7.
The previous claim 9 rejection under 35 U.S.C. 112(a) and 35 U.S.C. 103 has been withdrawn necessitated by cancellation of claim 9.
The previous claims 1-8 and 10-19 rejections under 35 U.S.C. 112(b) have been withdrawn necessitated by amendment of claims 1, 7, 8, 11-15, 17-19. However, new rejections were applied as described below.
Amendment of Claims (Rule 1.121)
Amendment of claims is non-compliant. Claim 9 was cancelled and text is present.
“When claim text shall not be presented; canceling a claim.
No claim text shall be presented for any claim in the claim listing with the status of "canceled." “ (MPEP 1.121)
However, in the interest of compact prosecution, the application is examined.
Claim Objections
Claim 1 is objected to because of the following informalities:
Applicant is suggested to replace “Obtain” with “Obtaining” in step (a) on line 7 of claim 1 to make it in line with “Dividing” in step b, “Purifying” in step c and so on.
Claim 14 recites: “is added the red blood cell fraction”. Applicant is suggested to replace recitation with: “is added to the red blood fraction”.
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.
Claims 1-8 and 10-19 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.
Regarding claim 1:
Claim 1 recites: “An in vitro method for rapid (less than five hours)”. “Rapid” is a relative term without a requisite standard taught in the specification and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Additionally, it is not clear if “less than five hours” in parenthesis is included in the scope of the claim or an optional limitation.
Claim 1 recites: “ an aquaculture-derived (sustainable), modified and activity-enhanced Limulus amebocyte lysate” in line 5 of page 1 and line 4 on page 2. “Sustainable” is a relative term without a requisite standard taught in the specification and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Additionally, it is not clear if “sustainable” is included in the scope of the claim since it is present in parenthesis. Claims 7, 12, 13, 18 and 19 have the same issue.
Step (b) recites: “incubating each aliquot with and without a panel of antibiotics and/or antifungal agents”. It is not clear how the same aliquot can be incubated with and without antibiotic and antifungal agents and whether intention was to aliquot sample and incubate different aliquots either with or without different antibiotics and antifungal agents.
Step (e) is directed to lysing the isolated red blood cells fraction. The isolation of the red blood cell fraction is described in step (c) and step (d) following step (c) is directed to washing of the isolated red blood cell fraction. It is not clear if step (e) is directed to lysing of isolated and washed red blood cell fraction or follows step (c) and is directed to lysis of isolated red blood cells without washing step.
Step (e) recites: “followed by a freeze and thawing cycle to yield a clarified lysate”. Since “clarified lysate” is not mentioned in the specification, it is not clear if “clarified lysate” is referred to lysate obtained after freeze and thawing cycle or an additional step is involved in clarification, e.g. centrifugation.
Claim 1 recites on p. 2, lines 18-19 that: “the detected organism is resistant (inhibition) or susceptible (acceleration), respectively, to the corresponding antimicrobial agent”. While it is clear that formation of a clot indicates the presence of Gram-negative bacteria and/or fungi and susceptibility of microorganism, e.g. bacteria, to antibiotic can cause destruction of the bacterial membrane and release of endotoxin and accelerate clot formation, it is not clear how inhibition of the clot formation can indicate resistant organism. If microorganism is resistant to antimicrobial agents, its amount will not significantly change and the clot will remain the same. Additionally, it is not clear why “(inhibition)” and “(acceleration)” are put in parenthesis, are these terms in parenthesis attempting to define the outcome or intending to limit the claim in another manner.
The scope and boundaries of claim 1 are not certain making claim 1 indefinite. Claims 2-8 and
10-19 do not resolve the issues mentioned above and are rejected.
Claim 7 recites: “substantially free of intact amebocytes”. “Substantially” is a relative term
which renders claim indefinite. The term “substantially” 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. It is not clear what amount of intact amebocytes is accepted to considered to be substantially free of intact amebocytes. The scope and boundaries of claim 7 are not certain making claim 7 indefinite.
Claim 13 has recites: “susceptibility testing of the gram-negative bacteria or fungi in the red blood cell fraction is determined by reduced or blocked formation of a self-supporting clot”. First, it is not clear how susceptibility testing can be performed in the red blood cell fraction which is an isolated fraction not containing microorganisms. Claim 1, from which claim 13 depends, recites incubation of specimen with antimicrobial agents in step (b) prior to isolation of the red blood cell fraction in step (c). Second, recitation of claim 13 contradicts claim 1 reciting acceleration of clot formation for susceptible microorganisms. It is not clear how susceptibility can cause reduction and blocking of clot formation within 60-120 minutes (limitation of claim 1) of incubation with antimicrobial agents. Acceleration of clot formation for susceptible to antibiotic microorganism is interpreted as caused by destruction of bacteria and release of endotoxin. Blocking of clot formation for susceptible microorganisms can occur as a result of reduction in the amount of microorganisms that will require longer culturing time and may not happen within 60-120 min incubation. The scope and boundaries of claim 13 are not certain making claim 13 indefinite.
Claim 13 is interpreted as directed to resistance of microorganism to antimicrobial agents not causing increase in the clot formation after incubation of red blood cell fraction isolated after incubation of specimen with the antibiotic with aquaculture-derived LAL.
Response to Arguments
Regarding 35 U.S.C. 112(b) rejection of claim 1 and more specifically the word “sustainable”, Applicant's argues (addressing p. 7, Comment 1 of the Remarks filed 05/27/2025) that claim recites: “aquaculture-derived, modified and activity-enhanced Limulus amebocyte lysate”. However, the claims still recites limitation “sustainable”, i.e.: “aquaculture-derived (sustainable), modified and activity-enhanced Limulus amebocyte lysate”. Applicant refers to the specification (paragraph 0031) describing modification and enhancement of activity of the Limulus amebocyte lysate. However, while the specification describes modification and enhancement of activity of LAL, the description of sustainable is not provided. Applicant further explains that: “… it was intended to convey that the aquaculture process permits repeated hemolymph collection from the same animals at frequent intervals, rendering the supply sustainable.” This explanation can be interpreted as referring to maintaining the certain level of supply due to aquaculture or supply with the same level of necessary ingredients since it comes from the same animal. The multiple interpretations of a claim limitation and the absence of support in the specification make the scope and boundaries of claims indefinite and additionally the parenthesis makes the scope of the claim uncertain since it is not clear if the word in parenthesis is a required limitation of the claim or optional limitation.
Amendment of claims overcomes the other 35 U.S.C. 112(b) rejections of claim 1 and rejections of claims 7, 11, 15 and 17. However, the 35 U.S.C. 112(b) rejection of claim 1 is maintained and new rejections are applied as described above.
Claim Rejections - 35 USC § 112
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.
Claims 1-8 and 10-19 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) contains 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 1 recites new limitations: “viscosity” and “viscometer” on p. 2, lines 9 and 13. This recitation is not supported by the specification.
Thus, since the support for the claim 1 is not provided, claim 1 contains new matter. One of ordinary skill in the art would not conclude that the applicant would have been in possession of the subject matter of claim 1 at the time of filing application.
Claims 2-8 and 10-19, dependent on claim 1, do not resolve the issues mentioned above and are rejected.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 2 and 12 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 2, dependent on claim 1, recites the limitation for the antibiotic or antifungal susceptibility testing determined in less than 5 hours. Claim 2 does not further limit claim 1 reciting “rapid (less than five hours) detection, differentiation and antibiotic or antifungal susceptibility testing”. Claim 2 does not recite any limitation that further limits method of claim 1.
Claim 12, dependent on claim 1, recites limitations for detection of gram-negative bacteria or fungi in the red blood cell fraction, i.e. formation of a self-supporting clot, incubation of red blood cell fraction with aquaculture-derived, modified and activity-enhanced LAL and period of incubation of 30 to 120 min at 37°C. These limitation are recited in claim 1. Claim 12 does not recite any limitation that further limits method of claim 1.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Maintained/Modified/New Rejections
The following rejections are maintained and/or modified taking into consideration amendment to claims filed on 05/27/2025.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-8, 10, 12-16 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Wainwright (US 20230258647 A1 filed 02/11/2022) in view of Tinker-Kulberg (Tinker-Kulberg et al. Front. Mar. Sci., 2020, 7, 541604, 1-19), Langevelde (Langevelde et al. Antimicrob. Agents and Chemother., 1998, 42, 739-743) and Whitney (Whitney, A Thesis, Greensboro, 2020).
Wainwright teaches detection and quantification of endotoxin in a sample with hybrid amebocyte lysate compositions (Abstract). The hybrid amebocyte lysate is composed of the native horseshoe crab amebocyte lysate which can be a Limulus polyphemus amebocyte lysate (LAL) and recombinant proteins, including zymogens and clotting factors (paragraphs 0014 and 0057). Wainwright discloses that the hybrid amebocyte lysate can be used for detection and differentiation of gram-negative bacteria and fungi depending on the composition: “Depending upon the components present in, or admixed with, the lysate it may produce a clot in the presence of an endotoxin, for example, a Gram negative bacterial endotoxin and/or a glucan, for example, a (1-3)-β-D glucan, produced by a yeast or a mold.” (paragraph 0057). Wainwright describes that the hybrid amebocyte lysate can have greater sensitivity in endotoxin detection compared to native amebocyte lysate (paragraph 0013).
Wainwright mentions that endotoxins can produce fever (paragraph 0003) and that the sample can be biological specimen of a body fluid: “ … body fluid to be tested for infection, including, for example, blood, lymph, urine, serum, plasma, ascites fluid, lung aspirants, and the like.” (paragraph 0131). Wainwright teaches mixing 0.1 ml of Limulus amebocyte lysate (LAL) reagent with 0.1 ml of a sample (paragraph 0154). The preferable temperature for the reaction is from 25° C to 40° C (paragraph 0109) and the time of the reaction is 30 min (paragraph 0204). Wainwright teaches detection of microbial contaminant (e.g. endotoxin) by hybrid amebocyte lysate using endpoint or kinetic assays with endpoint assays including endpoint chromogenic or turbometric assays (paragraph 0096). The product is detected visually or by using optical detector: “The method comprises contacting a hybrid amebocyte lysate (e.g., a hybrid amebocyte lysate disclosed herein) with a sample (e.g., a sample suspected of containing endotoxin), allowing the hybrid amebocyte lysate to react with the sample to produce a detectable product (e.g., a gel, increase in turbidity, or a colored or light-emitting product), and detecting the detectable product (e.g., visually or by the use of an optical detector).” (paragraph 0095).
The limitation “an opaque or non-opaque biological specimen” is interpreted as biological specimen having any opacity.
Wainwright does not teach the aquaculture-derived LAL, incubation of the biological specimen with antibiotic and/or antifungal agents, isolation, washing and lysis of red blood cells. Although Wainwright teaches detection of a gel visually, Wainwright does not describe determination of self-supporting gel by inverting the reaction vessel 180°. Although Wainwright teaches that all materials used for collection of specimen should be free of microbial contamination, Wainwright does not specifically teach obtaining biological sample with aseptic, sterile techniques.
Tinker-Kulberg teaches Limulus amebocyte lysate (LAL) obtained from a horseshoe crab aquaculture and optimization of the diet to improve biochemical properties of LAL (Abstract). Tinker-Kulberg discloses that the aquaculture-derived LAL has LAL activity higher than that of the commercial source which is a wild-type source (p. 6, right column, 4th paragraph, Figure 2, p. 16, left column, last paragraph) and the activity can be further increased with diet optimization reaching up-to 2.9 fold of the reference (p. 7, right column, 5th paragraph, Figure 4). Tinker-Kulberg describes that activity of LAL lysate is determined for the equal amount of protein in lysates (p. 5, right column, 2nd paragraph). Tinker-Kulberg mentions that the horseshoe crab aquaculture provides more “consistent LAL supply, eliminates mortality associated with wild-type bleeding practices and provides potential to enhance reagent quality and reactivity.” (p. 11, right column, 2nd paragraph).
Langevelde teaches antibiotic-induced LPS release from Salmonella typhii (Abstract). Langevelde describes incubation of bacteria with antibiotics at concentrations from 0.25 to 64 times MICs for 2 hours and measurement of bacteria killing and endotoxin, LPS, release (p. 740, right column, last paragraph, Figure 1). The release of LPS is determined by radioactivity associated with bacterial cell membrane containing LPS (p. 740, left column, 2nd paragraph). Figure 1 shows that 2 hour incubation demonstrates different susceptibility of S. typhi cells to two different antibiotics, ceftazidime and imipenem, and correlation between killing of bacteria and LPS release. Incubation with imipenem for 2h led the killing of a larger proportion of bacteria than incubation with ceftazidime (p. 740, right column, last paragraph) and imipenem induced 10-fold higher level of LPS release at identical concentrations (p. 741, left column, last paragraph). Thus, Langevelde teaching shows that susceptibility testing can be performed by detection of endotoxin release within 2 hour incubation of the sample aliquots with antibiotics.
Whitney teaches method of detection of bacteria and bacterial endotoxin in human blood using LAL assay (Abstract). Whitney discloses that hemoglobin of the red blood cells enhances activity of LPS and acts as an LPS binding protein (p. 37, 2nd paragraph). Whitney describes collection of blood using aseptic techniques and isolation of red blood cells by centrifugation at 2000 rcf for 10 min at 4° C, washing in isotonic 0.9% NaCl, resuspension, centrifugation and lysis (p. 18, 2nd paragraph). When bacteria are present in the blood, the samples are additionally subjected to freeze and thawing (p. 21, 1st paragraph). After sample preparation LAL is added to the blood sample and incubated at 37° C for 1 hour and after that gel-clot formation and clot integrity is visually assessed (p. 16, 2nd paragraph). Whitney describes that accession is performed by inverting the reaction tube 180° and samples forming gel-clots are able to withstand 180° conversion without movement of the clot (p. 26).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teachings of Tinker-Kulberg and Wainwright and substitute the LAL in Wainwright teaching with aquaculture-derived LAL from Tinker-Kulberg teaching in method of detection and quantification of endotoxin with hybrid LAL described by Wainwright. One would have been motivated to do so since Tinker-Kulberg teaches that horseshoe crab aquaculture provides consistent LAL supply, has higher LAL quality and reactivity compared to the wild-type source and has potential to further increase quality and activity. A skilled artisan would have reasonably expected success in the combination because Wainwright and Tinker-Kulberg teach Limulus amebocyte lysate for detection of gram-negative bacteria and endotoxin.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add teaching of Langevelde on evaluation of antibiotic susceptibility to the method of detection of gram-negative bacteria and fungi based on Wainwright and Tinker-Kulberg teachings and use 2 hour incubation of the specimen with antimicrobial agents as described by Langevelde and detect LPS by LAL assay as described by Wainwright and Tinker-Kulberg. One would have been motivated to do so since Langevelde showed different bacterial susceptibility to two types of antibiotics within 2 hour incubation based on endotoxin release. A skilled artisan would have reasonably expected success in the combination because Wainwright, Tinker-Kulberg and Langevelde teach detection of endotoxin.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that the increase in endotoxin release detected by Langevelde by radioactivity for susceptible antimicrobial agent will provide the increase or accelerate clot formation in LAL assay described by Wainwright and Tinker-Kulberg. One would be motivated to assume so with the reasonably expected success since LAL is shown by Wainwright and Tinker-Kulberg to produce clot in the presence of endotoxin and the increase in the amount of endotoxin will accelerate clot formation.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add method aseptic obtaining the blood specimen and isolation of red blood cells from the specimen described by Whitney or add red blood cells to the specimen lacking red blood cells and isolate the red blood cells fraction and then visually determine the clot formation by inverting the reaction vessel 180° and use that for method of detection of pathogens and antimicrobial susceptibility testing based on Wainwright, Tinker-Kulberg and Langevelde teachings. One would have been motivated to do so since Whitney teaches that hemoglobin of the red blood cells binds LPS and that can increase sensitivity of the assay and Whitney showed formation of the gel-clot after incubation of bacteria with the whole blood. A skilled artisan would have reasonably expected success in the combination because and Wainwright, Tinker-Kulberg and Whitney teach LAL assay for detection of endotoxin in specimen.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that detection, differentiation and antibiotic or antifungal susceptibility testing can be performed in less than five hours based on combination of Wainwright, Tinker-Kulberg, Langevelde and Whitney teachings. One would be motivated to assume so with the reasonably expected success since Langevelde teaches testing antibiotic susceptibility by incubating samples with antibiotics for 2 hours, Whitney describes isolation and lysis of the red blood cell fraction using rapid techniques and LAL test is performed for 1 hour as taught by Tinker-Kulberg (p. 5, right column, 2nd paragraph) and Whitney (p. 16, 2nd paragraph).
Thus, combination of teachings of Wainwright, Tinker-Kulberg, Langevelde and Whitney renders claim 1 obvious.
Regarding claim 2, as described in 112(d) rejection above, claim 2 does not further limit claim 1. Thus, teachings of Wainwright, Tinker-Kulberg, Langevelde and Whitney render claim 2 obvious.
Regarding claim 3, Langevelde teaches evaluation of antibiotic susceptibility without determination of the bacteria type. Thus, Langevelde teaching in combination with Wainwright, Tinker-Kulberg and Whitney teachings renders claim 3 obvious.
Regarding claim 4, Langevelde does not teach culturing or amplification of bacteria prior to the assay and hence Langevelde teaching in combination with Wainwright, Tinker-Kulberg and Whitney teachings renders claim 4 obvious.
Regarding claim 5, Wainwright teaches that biological specimen can be: “ … blood, lymph, urine, serum, plasma, ascites fluid, lung aspirants, and the like.” (paragraph 0131). Thus, Wainwright teaching in combination with Tinker-Kulberg, Langevelde and Whitney teachings renders claim 5 obvious.
Regarding claim 6, Wainwright teaches mixing 0.1 ml of Limulus amebocyte lysate (LAL) reagent with 0.1 ml of a sample (paragraph 0154). Thus, Wainwright teaching in combination with Tinker-Kulberg, Langevelde and Whitney teachings renders claim 6 obvious.
Regarding claim 7, Wainwright teaches the composition of the hybrid amebocyte lysate containing recombinant zymogens (factor B, factor C) and protein precursor (proclotting enzyme) with the amount higher compared to the native proteins: “ The composition comprises a horseshoe crab factor C, horseshoe crab factor B, and a horseshoe crab proclotting enzyme, wherein (a) the ratio of horseshoe crab factor B to horseshoe crab factor C is greater than the ratio of horseshoe crab factor B to horseshoe crab factor C in a native horseshoe crab amebocyte lysate, and/or (b) the ratio of horseshoe crab proclotting enzyme to horseshoe crab factor C is greater than the ratio of horseshoe crab proclotting enzyme to horseshoe crab factor C in a native horseshoe crab amebocyte lysate.” (paragraph 0009). Wainwright demonstrates in Example 2 that addition of recombinant factor B and recombinant proclotting factor increases the relative activity of the amebocyte lysate (paragraph 0156) and the increase is from 25% to about 100% as can be seen on Fig. 2. Wainwright mentions that the sensitivity of the hybrid amebocyte lysate can be at least 400% of the sensitivity of the reference native (wild-type) amebocyte lysate (paragraph 0052).
Tinker-Kulberg teaches the effect of diet on the biochemical properties of the aquaculture-derived LAL (Abstract). Tinker-Kulberg describes aquaculture-derived LAL on diet F providing 2.9-fold higher LAL activity than the reference, wild-type source, and higher amounts of factor C, factor B and proclotting enzyme (p. 8, right column, 6th paragraph, Figures 4, 7 and 8).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that combination of teachings of Tinker-Kulberg and Wainwright can provide at least 6-fold increase in the activity of LAL compared to the wild-type source. One would have been motivated to assume so with reasonably expected success since Wainwright teaches up-to 400% increase in the activity of the wild-type-derived LAL by addition of recombinant zymogens and clotting factors and Tinker-Kulberg teaches 2.9-fold increase in activity for diet-optimized aquaculture-derived LAL compared to the wild-type source. It can be envisioned that addition of recombinant zymogens and clotting factors to LAL from horseshoe crab aquaculture fed with the optimized diet can reach at least 6-fold increase in the activity of obtained LAL compared to the wild-type source. Thus, Wainwright, Tinker-Kulberg, Langevelde and Whitney teachings render claim 7 obvious.
Regarding claim 8, Whitney teaches development of a method for detection of LPS in biological samples by LAL test as described above. For that Whitney is using serial dilutions of LPS or bacteria in blood to represent the biological sample (Table 3.4 and Table 3.5). Thus, Whitney teaching in combination with Wainwright, Tinker-Kulberg and Langevelde teachings renders claim 8 obvious.
Regarding claim 10, Whitney teaches purification of the red blood cells from the sample by centrifugation at 2000 rcf for 10 min at 4° C (p. 18, 2nd paragraph). Thus, Whitney teaching in combination with Wainwright, Tinker-Kulberg and Langevelde teachings renders claim 10 obvious.
Regarding claim 12, Whitney teaches incubation of LAL with the blood sample LPS and bacteria containing LPS for 1 hour at 37° C (p. 16, 2nd paragraph). Thus, Whitney teaching in combination with Wainwright, Tinker-Kulberg and Langevelde teachings renders claim 12 obvious.
Regarding claim 13, as described above, claim 13 is interpreted as directed to resistance of microorganism to antimicrobial agents not causing increase in the clot formation after incubation of red blood cell fraction isolated after incubation of specimen with the antibiotic with aquaculture-derived LAL as described for 112(b) rejection above. Langevelde teaches resistance of S. typhi to ceftazidime at concentrations up-to 1 µg/mL (p. 740, Figure 1) since the number of bacteria were not significantly reduced and the LPS released did not significantly increased.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that the absence of the increase in LPS release detected by Langevelde by radioactivity for resistant to ceftazidime bacteria will not provide increase of clot formation in LAL assay described by Wainwright and Tinker-Kulberg. One would be motivated to assume so with the reasonably expected success since LAL is shown by Wainwright and Tinker-Kulberg to produce clot in the presence of endotoxin and in the absence of changes in LPS level the clot formation will not increase. Thus, Wainwright, Tinker-Kulberg, Langevelde and Whitney teachings render claim 13 obvious.
Regarding claim 14, Whitney teaches that hemoglobin of the red blood cells enhances activity of LPS and acts as an LPS binding protein (p. 37, 2nd paragraph). Whitney describes that during detection of bacteria with the LAL assay, bacteria were added and preincubated with the whole blood for 15 minutes prior to isolation of red blood cells (p. 21, last paragraph) resulting in gel-clot formation (Figure 3.14). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Whitney guidance and add whole blood to the sample other than blood. One would have been motivated to do so since Whitney teaches that hemoglobin of the red blood cells binds LPS and that can increase sensitivity of the assay. A skilled artisan would have reasonably expected success in the modification because Whitney showed formation of the gel-clot after incubation of bacteria with the whole blood and Wainwright and Tinker-Kulberg provided methods of detection of pathogen by the LAL assay. Thus, Whitney teaching in combination with Wainwright, Tinker-Kulberg and Langevelde teachings renders claim 14 obvious.
Regarding claim 15, Wainwright teaches a kit for bacterial endotoxin testing comprising recombinant factor C, factor B and pro-clotting enzyme (claim 65). Thus, Wainwright teaching in combination with Tinker-Kulberg and Langevelde and Whitney teachings renders claim 15 obvious.
Regarding claim 16, Wainwright teaches that the multi-step kinetic LAL assay can be run in a format of a cartridge, comprising compartments for the hybrid amebocyte lysate, sample and a chromogenic or fluorogenic substrate and pumps for fluid communication (paragraphs 0102-0104). Wainwright discloses that the assay can be performed in well-type format with automated addition of reagents: “In the well-type format, the samples and reagents are added to each of the wells, preferably using an automated system, such as a robot, and the plate processed by a microplate reader, which can be programmed to sequentially read the absorbance of each well in a repetitive fashion.” (paragraph 0107). Thus, Wainwright teaching in combination with Tinker-Kulberg, Langevelde and Whitney teachings renders claim 16 obvious.
Regarding claim 18, Wainwright teaches that amebocyte lysate can be derived from different horseshoe crabs: “Preferred amebocyte lysates can be derived from horseshoe crabs belonging to the Limulus genus, for example, Limulus polyphemus, the Tachypleus genus, for example, Tachypleus tridentatus and Tachypleus gigas, and the Cacrinoscorpisus genus, for example, Cacrinoscorpisus rotundicauda. (paragraph 0057). Wainwright further discloses that for hybrid amebocyte lysate any horseshoe crab factor B or proclotting enzyme can be used: “any horseshoe crab factor proclotting enzyme, for example, a Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas, or Cacrinoscorpisus rotundicauda factor B, may be used in the practice of the invention.” (paragraph 0063) and “any horseshoe crab factor proclotting enzyme, for example, a Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas, or Cacrinoscorpisus rotundicauda proclotting enzyme, may be used in the practice of the invention.” (paragraph 0072).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that the horseshoe crab belonging to the Tachypleus genus and/or the Cacrinoscorpisus genus can be cultured in aquaculture similar to Limulus horseshoe crab as described by Tinker-Kulberg and that the hybrid LAL based on Wainwright and Tinker-Kulberg teachings can further comprise aquaculture-derived Tachypleus amebocyte lysate or Cacrinoscorpisus amebocyte lysate. One would be motivated to assume so with the reasonably expected success since Wainwright teaches that amebocyte lysate can be derived from different horseshoe crabs and it is within the skills of artisan in the field to apply aquaculture technique developed by Tinker-Kulberg for Limulus horseshoe crab to Tachypleus or Cacrinoscorpisus horseshoe crabs. Thus, Wainwright, Tinker-Kulberg, Langevelde and Whitney teachings render claim 18 obvious.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Wainwright (US 20230258647 A1 filed 02/11/2022) in view of Tinker-Kulberg (Front. Mar. Sci., 2020, 7, 153, 1-13), Langevelde (Langevelde et al. Antimicrob. Agents and Chemother., 1998, 42, 739-743) and Whitney (Whitney, A Thesis, Greensboro, 2020) as applied to claim 1 above, and further in view of Yung (WO 2011091037 A2).
The teachings of Wainwright, Tinker-Kulberg, Langevelde and Whitney have been set forth above.
Wainwright, Tinker-Kulberg, Langevelde and Whitney do not teach purification of the biological specimen using microbeads or magnetic nanospheres.
Yung teaches magnetic microbeads that have an affinity to bind target components, such as pathogens in the fluid. Magnetic field gradient provides rapid separation of bound components that can be further analyzed using various techniques (Abstract). Yung discloses that the source fluid can be whole blood in which pathogens can be detected (paragraph 0012). Yung describes that magnetic beads can be coated with molecules specifically binding the surface of pathogenic cell in complex fluid, such as whole blood (paragraph 0066). Yung teaches that magnetic beads can be coated with mannose binding lectin that recognizes surface markers on a wide variety of pathogens (paragraph 0088). Yung describes the magnetic field strength in the range of 0.1 Tesla to 1 Tesla (paragraph 0074).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add separation of pathogens using magnetic microbeads as described by Yung to method of detection, differentiation and susceptibility testing of pathogens based on Wainwright, Tinker-Kulberg, Langevelde and Whitney teachings. One would have been motivated to do so to increase detection sensitivity since after separation of pathogens other components of biological fluid will not interfere with the assay and to reduce detection time because separation of pathogens will provide sufficient pathogen concentration not requiring additional culturing. A skilled artisan would have reasonably expected success in the combination because Yang provides method of rapid purification of pathogens from the biological fluid and Wainwright, Tinker-Kulberg, Langevelde and Whitney teach tests for detection and differentiation of pathogens. Thus, Yung teaching in combination with Wainwright, Tinker-Kulberg, Langevelde and Whitney teachings renders claim 11 obvious.
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Wainwright (US 20230258647 A1 filed 02/11/2022) in view of Tinker-Kulberg (Front. Mar. Sci., 2020, 7, 153, 1-13), Langevelde (Langevelde et al. Antimicrob. Agents and Chemother., 1998, 42, 739-743) and Whitney (Whitney, A Thesis, Greensboro, 2020) as applied to claim 1 above, and further in view of Das (Das et al. Biosensors and Bioelectronics, 2014, 51, 62-75).
The teachings of Wainwright, Tinker-Kulberg, Langevelde and Whitney have been set forth above.
Wainwright, Tinker-Kulberg, Langevelde and Whitney do not teach techniques of claim 17 for analysis of biological specimen.
Das teaches different techniques for detection of endotoxin (Abstract). Das describes application of the LAL method and its mechanism (p. 70, right column, p. 71, p. 72, left column, 1st paragraph, Fig, 9 and Fig. 10). Das teaches multiple techniques other than LAL test including PCR and ELISA and describes characteristics of the techniques, e.g. detection limit (p. 65, right column, last paragraph, p. 66, left column, 1st paragraph, Table 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow guidance of Das and add techniques other than LAL test for detection, differentiation and susceptibility testing of pathogens based on Wainwright, Tinker-Kulberg, and Whitney teachings. One would have been motivated to do so to support and verify results of LAL testing because Das describes characteristics of multiple techniques including the detection limits allowing to select technique suitable for the application. A skilled artisan would have reasonably expected success in the combination because Das, Wainwright, Tinker-Kulberg and Whitney teach LAL tests for detection of endotoxins and Das provides other techniques for endotoxin detection. Thus, Das teaching in combination with Wainwright, Tinker-Kulberg, Langevelde and Whitney teachings renders claim 17 obvious.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Wainwright (US 20230258647 A1 filed 02/11/2022) in view of Tinker-Kulberg (Front. Mar. Sci., 2020, 7, 153, 1-13), Langevelde (Langevelde et al. Antimicrob. Agents and Chemother., 1998, 42, 739-743) and Whitney (Whitney, A Thesis, Greensboro, 2020) as applied to claim 1 above, and further in view of Inada (Inada et al. Microbiol. Immunol., 2003, 47, 701-707).
The teachings of Wainwright, Tinker-Kulberg, Langevelde and Whitney have been set forth above.
Wainwright, Tinker-Kulberg, Langevelde and Whitney do not teach LAL to further comprise Bombyx mori hemolymph for detection of gram-positive bacteria.
Inada teaches the silkworm (Bombyx mori) larvae plasma (SLP) test for diagnosis of bacterial meningitis in patients (Abstract). Inada describes that SLP test is based on the melanin formation in the presence of peptidoglycan present in the cell walls of gram-positive and gram-negative bacteria (p. 701, left column). The SLP test was used together with LAL test: “When this test was used together with two types of limulus tests, an endotoxin-specific test, and a conventional test, meningitis was further characterized as gram-positive, gram-negative or fungal meningitis.” (Abstract). Inada discloses that SLP test can be performed by quantitative colorimetric measurement or by visually observing the darkening color for the simple and rapid diagnosis of meningitis (Abstract). The combined test allowed to determine source of infection for meningitis: “ … we were able to discriminate between meningitis caused by gram-negative bacteria, gram-positive bacteria, and fungi by using a combination of the SLP and limulus tests” (p. 701, right column).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add teaching of Inada to teachings of Wainwright, Tinker-Kulberg, Langevelde and Whitney and combine SLP and LAL tests for detection, differentiation and susceptibility testing of pathogens described by Wainwright, Tinker-Kulberg and Whitney teachings. One would have been motivated to do so since Inada teaches that SLP test can determine gram-positive bacterial infection and combination of SLP and LAL tests will allow to discriminate between gram-negative bacteria, gram-positive bacteria, and fungi causing the disease and hence provide appropriate treatment. A skilled artisan would have reasonably expected success in the combination because Inada, Wainwright, Tinker-Kulberg and Whitney teach LAL tests that can detect gram-negative bacteria and fungi and Inada teaching provides additional test for gram-positive bacteria and method to discriminate between different types of infection. Thus, Inada teaching in combination with Wainwright, Tinker-Kulberg, Langevelde and Whitney teachings renders claim 19 obvious.
Response to Arguments
Applicant's arguments filed 05/27/2025 have been fully considered but they are not persuasive.
Applicant’s arguments with respect to combination of the prior art of Wainwright, Chmelar and Whitney are moot because the current rejection is modified necessitated by amendment of claims and is based on combination of the prior art of Wainwright, Tinker-Kulberg, Langevelde and Whitney as described above, where Wainwright teaches the improved hybrid amebocyte lysate composed of the wild-type LAL and recombinant zymogens and proclotting factors, Tinker-Kulberg describes the optimized aquaculture-derived LAL with enhanced activity compared to the wild-type-derived LAL which can be combined with Wainwright LAL, Langevelde provides the susceptibility testing method and Whitney describes isolation of red blood cells for endotoxin detection and all of them describe different aspects of the method for detection, differentiation and antimicrobial susceptibility testing as described in the rejection above.
In response to Applicant’s arguments (addressing pages 9-10 of the Remarks) that: “That different scientific principle yields unexpected performance: detection limits drop from 0.5 EU mL-1 to 0.05 EU mL-1; lot-to-lot variability disappears; and reaction time is reduced from 60 min to 30 min.”, these arguments are not persuasive because:
The current rejection includes prior art of Tinker-Kulberg teaching horseshoe crab
aquaculture that eliminates lot-to-lot variability of commercial preparations. The prior art of Wainwright does teach the reaction time for LAL assay of 30 min (paragraph 0204). Wainwright describes detection of endotoxin at as low as 0.0156 EU/ml by clot formation assay when using hybrid LAL lysate (Figure 5B, paragraph 0170). Thus, the described features are not unexpected by the prior art.
The described features are not supported by the specification because the specification
does describe detection limits and does not describe reaction time lower than 60 min. Additionally, claims are not commensurate in scope with the unexpected results. MPEP 716.02: “Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In other words, the showing of unexpected results must be reviewed to see if the results occur over the entire claimed range.” In instant case, claims do not have limitation for the detection limits and the lot-to-lot variability and the limitation for the reaction time is 60 min and does not include 30 min. Therefore, since these limitations are not present in the claims, the claims are not commensurate in scope with the unexpected results.
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 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.
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/L.G.K./Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653