Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 103
The 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.
Claim(s) 1, 2, 3-6, 17-19, 21, 23, 25 and 37-40 are rejected under 35 U.S.C. 103 as being unpatentable over CN 103675115 (hereinafter “Zhang”) in view of JP 2004309189 (hereinafter “Imagawa”) and further in view of US 20190086306 (hereinafter “Lopez”).
Zhang teaches the following.
The invention relates to natural medicine screening technology field, the invention claims a marine natural active substance bead high-content quick screening method, is based on bead bond and high connotation target protein integrated active substance screening and evaluation method, the arrangement of the invention are: the hardware system is a permanent magnet, a magnetic bead, a target software system responsible for hygroplasm data collection. Compared with the screening method and the traditional ultraviolet screening method of the invention, obviously improves the screening efficiency, shorten the filtering time, and meanwhile able to inspect the multiple biological activities, discovery of a synergistic effect between the compound. screening method of the invention is quick and accurate, large information quantity and good repeatability, which can be used for large-scale screening natural product library or a combinatorial chemical library. See abstract.
The invention is directed to screening a target, comprising the following steps (para. 0007):
A, using magnetic bead as carrier, functionalized to bind to a target (para. 0008)
B, incubating the magnetic bead with a sample (para. 0009)
C, removing the supernatant, washing the magnetic beads by denaturing solvent to denature the enzyme, and collecting the eluent (para. 0010);
identifying a substance in the eluent through liquid phase chromatography-mass spectrometry and nuclear magnetic resonance technology authentication analyzing, obtaining target protein inhibitor, wherein the liquid chromatography-mass spectrometry (hygroplasm) parameter is ESI positive and negative ion mode (para. 0011);
said target protein is a DNA topoisomerase, alpha-glucosidase or triglyceride enzyme and so on (para. 0012)
in said step A is specifically as follows: the magnetic bead washing several times, adding 1 - (3 amino propyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activated magnetic bead surface, adding dissolved DNA topoisomerase, alpha-glucosidase and triglycerides such as drug-related target protein, after incubating for a period of time, the enzyme is bonded to the magnetic bead surface, cleaning for several times (para. 0013).
The sample comprises a marine organism (para. 0014).
The screening method of the invention includes providing
a concentration of 50 mg/mL with 25mM MES (pH 6) is 50 mg/mL N-hydroxy succinimide (NHS) solution. on the magnetic beads after cleaning, adding 50 microlitres of EDC solution and 50 microlitres of NHS solution, uniformly mixing, slowly inclining rotating at room temperature hatching for 30 min. after incubation, putting the centrifugal tube absorbing 4 min on the magnet, removing the supernatant, using 300 mu L 25mM MES (pH6) washing for two times (para. 0019);
second step: on the activated magnetic bead, respectively adding 60 mu L dissolved in 25mM MES solution (pH DNA topoisomerase 6) 1, alpha-glucosidase and triglyceride, and respectively adding 40 mu L 25mM MES solution (pH 6) to a final volume of lOOyL, oscillating and mixing uniformly at room temperature incubation overnight, after incubation, putting the centrifugal tube absorbing 4 min on the magnet, removing the supernatant; using 300 mu L PBS coated bead 4, covers proteins such as bovine serum albumin (BSA) or skim milk powder concentration can reach 0.1-0.5 %, the coated beads dispersed in the required concentration of PBS and Tris buffer solution again (para. 0021);
the third step: using the transmission electronic microscope to blank beads and bonded bead, characterizing the properties [para. 0022];
a fourth step: weighing marine natural product extract, adding a small amount of dimethyl sulfoxide (DMSO) to dissolve, then adding
0.1 mL phosphate buffer saline (PBS) solution (pH 6.8, 10mM) ultrasonic IOmin to dissolve, absorbing supernatant liquid mass analysis to obtain HP(para. 0023).LC and molecular quantity information
fifth step: fourth step marine natural product to obtain supernatant 40 μ L, respectively adding to 100 mu L, DNA topoisomerase 1 alpha-glucosidase and triglyceride enzyme beads dispersed in acetic acid solution and blank beads dispersed in the PBS buffer solution, placing on the oscillator mixing uniformly, incubated for I hour, using PBS buffer solution to wash for 3 times, each time of cleaning solution in liquid phase analysis (para. 0024);
a sixth step of removing the cleaning liquid, each connected in the container adding 10% acetonitrile-water (mL) to wash eluent to liquid-mass spectrometry analysis (para. 0025);
seventh step: through comparing the fourth step and the sixth step liquid obtained by mass analysis spectrum can be obtained with DNA topoisomerase 1, alpha-glucosidase and bead triglyceride binding of marine natural product retention time and molecular quantity information (para. 0026).
An instrument is disclosed for high performance liquid chromatography tandem dbrs LCQ Deca XPplus ion trap mass spectrometry, chromatographic column: Agilent Zorbax SB-C18 column (4.6X 250 mm, 5 microns) ;Agilent 1200 preparation liquid phase (XDB-C18column, 30X, 250 mm, 5 microns); constant temperature oscillation incubator. ELx800 microplate reader. NMR (Bruker AvanceIII 500M NMR). (Paras. 0044-0045).
Embodiment 1: key based on magnetic beads and DNA topoisomerase, high-content screening method of alpha-glucosidase and triglyceride (para. 0046) on the activated magnetic beads, respectively adding 60 mu L dissolved in 25mM MES solution (pH 6) of DNA topoisomerase Ka-glucosidase and triglyceride, and respectively adding 40 mu L 25mM MES solution (pH 6) to a final volume of 100 [mu] L, oscillating and mixing uniformly at room temperature incubation overnight, after incubation, the tube is placed on the magnet 4 min and removing the supernatant. using 300 mu L PBS coated bead 4, covers proteins such as BSA or skim milk powder concentration can reach 0.1-0.5 %, the coated beads dispersed in the required concentration of PBS and Tris buffer solution again (para. 0048).
Using a transmission electron microscope to the blank bead to bead and bonding property characterization, can be seen from FIG. 1, clear of blank beads (A) under the transmission electron microscope, and combines the bead of the triglyceride (B) surface is very fuzzy, which means it has a protein adhesion on the magnetic bead surface (para. 0049).
Thus, as to Applicant’s claim 1, Zhang teaches:
a method of preparing a protein analyte for characterization [see abstract, paras. 0007, 0011, 0023, 0025-0026]
comprising pretreating a protein analyte that includes providing a denaturing solvent to denature the protein in the presence of the first magnetic beads [abstract, paras. 0010 and 0012]
However, while Zhang teaches oscillating and mixing the beads and reagents, Zhang does not teach that the pretreatment is done on magnetic beads in an oscillating magnetic field to disaggregate the magnetic beads and stir an aqueous solution [that is, Zhang does not teach use of a magnetic field to oscillate], nor that the analyte to be analyzed is the protein that was bound to the magnetic beads.
Zhang however does show that it is known in the art that proteins can be bound magnetic beads and that proteins can be pretreated with denaturing solvent to denature a protein/enzyme [even though Zhang teaches analysis of the protein’s inhibitor.]
Moreover, it is well understood in the art that an analyte to be analyzed can be a protein bound to magnetic beads, as may be desirable for study. It is also well known in the art that the magnetic beads, and therefore the bound protein to be detected, can be manipulated, by a magnetic field. It is predictable to the ordinary artisan that the protein analyte can be released from magnetic beads and detected according to the selected analysis technique.
Furthermore, the teachings of Imagawa however suggest the missing limitations.
Imagawa discloses a method wherein an active component specifically reacting with the specific component is used. A magnetic field is applied to the magnetic fine particles [immobilized with the active component], and rotating and / or migrating the magnetic fine particle clusters in the medium [is performed] by applying an oscillating magnetic field to promote the reaction between the specific component and the active component. The oscillating magnetic field is in the range of 1 to 1000 Hz. A method for accelerating a reaction is thereby provided. Para. 0010.
Imagawa teaches a method for accelerating a reaction of a specific component in a sample medium, and forming a magnetic particle cluster by applying a magnetic field to magnetic particles immobilized with an active component that specifically reacts with the specific component. Then, by applying an oscillating magnetic field, the magnetic fine particle cluster is rotated and / or migrated in the medium to obtain a stirring effect, thereby shortening the reaction time. Para. 0013.
It would have been obvious to one skilled in the art to modify Zhang to provide magnetic particles in an oscillating magnetic field to promote reaction in the Zhang invention, as taught by Imagawa. Examiner notes that such oscillation magnetic field would disaggregate the magnetic beads and cause the magnetic beads to stir an aqueous solution in the Zhang invention.
Also, as to claim 1, Zhang also does not disclose detecting a pretreated protein [i.e., a denatured protein] (as in Applicant’s claim 1), nor that the pretreatment is with a chaotropic agent, (as in Applicant’s claim 3).
Lopez teaches the following, which discloses that it was known in the prior art that a protein to be detected can be denatured by chaotropic agents and digested with enzymes for analysis by mass spectrometry.
Lopez teaches a method for preparing a protein-containing sample for analysis by mass spectrometry includes introducing the sample into a reaction vessel. The reaction vessel contains a reagent mixture including pre-measured quantities of an immobilized proteolytic enzyme, a reducing agent and an alkylating agent. See abstract and paras. 0006-0008.
Lopez teaches a method for preparing a protein-containing sample for mass spectrometry analysis, comprising: concurrently reacting the sample with an alkylating agent, a reducing agent and an immobilized proteolytic enzyme; and activating the sample by performing at least one of heating the sample, sonicating the sample, and applying pressure to the sample. Para. 0009.
The term “alkylating efficiency” is defined as the quantitative relation between the number of detected cysteines that have been modified by the alkylating agent during the reaction and the total number of cysteines detected in the sample, as determined by analyzing the sample using mass spectrometry followed by a database search. A specific and non-limiting example includes alkylation of cysteine with chloroacetamide (CAA) or Iodacetic acid (IAC) that increases the mass of the peptide by 57.021 or 58.005 Daltons, respectively. This increase in mass is measured using a mass spectrometer and quantified during the database search. Para. 0028.
Referring now to FIG. 1A, shown is an overview of the workflows of three prior art methodologies for preparing a protein-containing sample for subsequent analysis by mass spectrometry, as well as the workflow of a new one-step protocol according to an embodiment. The so-called “classic method” includes the steps of cell lysis, protein abundance determination,
denaturation/reduction/alkylation, and digestion, all of which are performed sequentially. In the cell lysis step a lysis buffer solution, containing salts such as Tris-HCl or EDTA and optionally detergents such as Triton X-100 or SDS, is added to the sample to break open the cells contained therein. The “protein abundance” step provides a quantitative measure of protein in the sample, so that the concentration of trypsin used for the subsequent digestion step may be adjusted to yield a trypsin-to-protein ratio of about 1:50 (wt:wt). The proteins are then denatured with strong chaotropic agents such as urea or thiourea. This step is either followed by or combined with disulfide reduction using a reducing agent such as tris(2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DTT). The free sulfhydryl groups on the cysteine residues are then alkylated with reagents such as iodoacetamide or iodoacetic acid to irreversibly prevent the free sulfhydryls from reforming disulfide bonds. The denatured, reduced and alkylated proteins are then digested by endoproteinases, (e.g., trypsin, chymotrypsin, Glu-C and Lys-C), which hydrolytically break peptide bonds to fragment proteins into peptides. The last two steps of the classic method are particularly time-consuming, accounting for most of the six-and-a-half to twenty-and-a-half hours required to complete a sample preparation. Para. 0031.
Referring now to FIG. 1B, the “Classic Method” workflow is shown in more detail. Each step that was discussed with reference to FIG. 1A comprises a series of sub-steps, each requiring various reagent additions and/or other user intervention. The cell lysis step includes the addition of a lysis buffer and the addition of benzoate, after which sonication occurs. The protein concentration in the resulting solution is then determined in a separate step. Next, a protein denaturation step is performed by adding a reducing agent and an alkylating agent. The duration of the protein denaturation step is about 2 hours. Finally, a digestion step is performed by adding Lys C, usually at a 1:100 enzyme:substrate ratio, and incubating for about 2 hours, after which a buffer is added and trypsin is added, in this case the enzyme:substrate ratio tend to vary from 1:10 up to 1:100, and the sample is incubated again for an additional 4 to 16 hours. After an acid addition step to stop the enzymatic activity, the sample is ready to be cleaned up and analyzed by mass spectrometry. Para. 0033.
Referring now to FIG. 1C, the two-step protocol according to an embodiment is shown in more detail. Samples that need an extra energy input during the protein extraction process are e.g., processed using the two-step protocol described below. In step 1 of the two-step protocol the sample is added to a sonication tube containing a lysis buffer. The lysis buffer contains a buffered solution, e.g. ammonium bicarbonate 50 mM pH 8.2 and suitable chaotropic agents or detergents that are capable of disrupting the hydrogen bonding network between water molecules and reducing the stability of the native state of proteins and membranes by weakening the hydrophobic effect. Usually, 6M urea, 1% SDS, 1% SDC are the typical additives. Once the lysis buffer has been added to the sample, the resulting solution is sonicated for e.g., about 5 minutes. High intensity ultrasound energy helps disrupt DNA as well as cell membranes, allowing proteins to be extracted efficiently from cellular matrices of the sample. In step 2 of the two-step method, the lysis buffer containing the sample is added to a reaction tube containing a reagent mixture including pre-measured quantities of immobilized proteolytic enzymes, a reducing agent, and an alkylating agent. By way of a specific and non-limiting example, 120 uL of immobilized trypsin slurry is added to the vessel, the beads are cleaned three times with ammonium bicarbonate, and then the supernatant is removed from the tube. A solution containing 5 mM of TCEP, 15 mM CAA in 50 mM ammonium bicarbonate pH 8.2 is then added to the beads and the mixture is ready for the sample to be added. Once the sample is added, the tube contents are heated at e.g., 50° C. for an additional e.g., 60 minutes. The tube or vessel can be centrifuged or the solution filtered out to remove the beads, after which the sample is ready to be analyzed by mass spectrometry. Para. 0034.
Referring now to FIG. 1D, the one-step protocol according to an embodiment is shown in more detail. In step 1 of the one-step protocol the user adds the sample into a reaction vessel containing a reagent mixture including a lysis buffer, pre-measured quantities of an immobilized proteolytic enzyme, a reducing agent and an alkylating agent. The contents of the reaction vessel are then activated, such as for instance by sonication for a period of about 5 minutes or by heating between 37° C. and 50° C. for between five and ninety minutes. As used herein, the term “activating” and its cognates refers to the act of adding energy to the contents of the reacting vessel so as to accelerate the rate of the desired reaction of the sample (or components thereof) with the various reagents. This workflow is envisioned for use with, for instance, mammalian cells and other types of samples such as purified proteins where exhaustive protein extraction is not needed, and the ultrasound step can be skipped during the protein extraction step. Para. 0035.
A specific and non-limiting example of the one-step protocol according to an embodiment uses a closeable reaction vessel, such as for instance an Eppendorf tube, which contains a reagent mixture prepared using 1 μL tris(2-carboxyethyl)phosphine (TCEP) 0.2 M solution, 0.2 mg of CCA, 0.8 mg of CaCl2 and 50 μL Poroszyme® immobilized trypsin beads. In this example, the reagent mixture includes a proteolytic enzyme in the form of immobilized trypsin (such as Poroszyme® beads, available from Thermo Fisher Scientific), however, other suitable immobilized enzymes may be used, as long as the immobilization process helps to stabilize the secondary and tertiary structure of the protein without decreasing its activity. Importantly, having the enzyme immobilized to a solid support allows the enzyme to be present in a very high enzyme:substrate ratio (i.e. 100:1), forcing the enzyme kinetics to speed up due to its dependence on enzyme concentration. Advantageously, the immobilized enzyme does not contaminate the sample and it can easily be removed from the sample, since the solid support is very easy to separate from the solution. A non-limiting list of alternative reagents and/or concentrations includes at least the following: buffer pH: 7.8-8.2; reducing agent TCEP 1-5 mM; alkylating agent CAA 10 mM-25 mM; alkylating agent IAC 1-5 mM; alkylating agent IAA 1-15 mM; and CaCl.sub.2 0.1-1 mM. As suggested above, the reagent mixture optionally includes a proteolytic enzyme other than trypsin, such as for instance one of LysC, LysN, AspN, GluC, ArgC or chymotrypsin. Optionally, the reagent mixture is dried down and is stored below room temperature prior to use, such as for instance stored at 4° C., −20° C. or −80° C. Para. 0036.
In certain embodiments of the invention, the reagent mixture contained within the prepared reaction vessel includes an isobaric labeling reagent that reacts with the peptides formed by protein digestion to produce isotopically labeled peptides. For example, a kit prepared in accordance with an embodiment may include a set of reaction vessels containing the above-described reagent mixture (comprising reducing and alkylating agents and immobilized proteolytic enzyme), each one of which further contains a different isotopologue of an isobaric labeling reagent, such that differentially labeled samples may be prepared and subsequently combined so as to determine relative quantities of peptides of interest via analysis by liquid chromatography-mass spectrometry, using techniques well known in the art. Isobaric labeling reagents are available from Thermo Fisher Scientific under the trade name Tandem Mass Tags. Para. 0037.
The stability of the reaction mixture that is employed in the new protocol, which includes immobilized trypsin, a reducing agent (TCEP), and an alkylating agent (CAA), was investigated and the results are presented in FIGS. 8-10. More particularly, the reagent mixture was prepared and then stored at various temperatures between −80° C. and 24° C. for a period of three weeks, prior to being used to incubate samples containing one million HELA cells. The HELA cells were added to previously stored reaction mixture, and digested using the new protocol according to an embodiment of the invention (i.e., with heating for 1 h @50° C.). After the digestion, lug of digested peptides were transferred to an autosampler vial, and then analyzed by LC-MS/MS using a 2 h gradient in a Thermo Scientific EASY-nLC1200 system coupled to a Thermo Scientific Q Exactive HF mass spectrometer. Para. 0053.
Thus, regarding Applicant’s claims 1, 3-6, 21, 23 and 25, the above disclosures show that Lopez teaches and/or suggests the following limitations as part of a method for preparing a protein-containing sample for analysis by mass spectrometry:
providing a chaotropic agent to protein analyte in an aqueous solution to denature the protein (as in Applicant’s claim 3);
providing a reducing agent to the denatured protein solution to provide a reduced, denatured protein analyte (as in Applicant’s claim 4);
mixing to expose the denatured protein analyte to an alkylating agent to provide
an alkylated protein analyte (as in Applicant’s claim 5);
quenching excess alkylating agent by adding a reducing agent to the alkylated protein analyte to provide the pretreated protein analyte (as in Applicant’s claim 6);
wherein the chaotropic agent is an organic chaotropic agent (as in Applicant’s claim 21, 23);
wherein the alkylating agent is an iodoacetic acid (as in Applicant’s claim 25); and
wherein pretreating and enzymatically digesting are performed sequentially (as in Applicant’s claim 28).
In the modification of the teachings of Zhang as discussed further above, it would have been obvious to one skilled in the art to utilize the reagents and steps disclosed by Lopez, including denaturing the proteins with strong chaotropic agents (para. 0031) for protein analysis by mass spectrometry, since Lopez teaches that these are known and classic methods for preparing a protein-containing sample for analysis by mass spectrometry.
In summary, as to Applicant’s claim 1, Zhang teaches:
a method of preparing a protein analyte for characterization [see abstract, paras. 0007, 0011, 0023, 0025-0026]
comprising pretreating a protein analyte that includes providing a denaturing solvent to denature the protein in the presence of the first magnetic beads [abstract, paras. 0010 and 0012]
However, while Zhang teaches oscillating and mixing the beads and reagents, Zhang does not teach that the pretreatment is done on magnetic beads in a changing magnetic field to disaggregate the magnetic beads and stir an aqueous solution [that is, Zhang does not teach use of a magnetic field to oscillate], nor that the analyte to be analyzed is the protein that was bound to the magnetic beads.
Zhang however does show that it is known in the art that proteins can be bound magnetic beads and that proteins can be pretreated with denaturing solvent to denature a protein/enzyme [even though Zhang teaches analysis of the protein’s inhibitor.]
However, it is well understood in the art that an analyte to be analyzed can be a protein bound to magnetic beads, as may be desirable for study. It is also well known in the art that the magnetic beads, and therefore the bound protein to be detected, can be manipulated, by a magnetic field. It is predictable to the ordinary artisan that the protein analyte can be released from magnetic beads and detected according to the selected analysis technique.
Moreover, it would have been obvious to one skilled in the art to modify Zhang to provide magnetic particles in an oscillating magnetic field to promote reaction in the Zhang invention, as taught by Imagawa. Examiner notes that such oscillation magnetic field would disaggregate the magnetic beads and cause the magnetic beads to stir an aqueous solution in the Zhang invention.
Furthermore, Lopez teaches providing a chaotropic agent to denature a protein and to enzymatically digest the protein before analysis by mass spectrometry.
In the modification of the teachings of Zhang as discussed further above, it would have been obvious to one skilled in the art to utilize the reagents and steps disclosed by Lopez, including denaturing the proteins with strong chaotropic agents (para. 0031) for protein analysis by mass spectrometry, since Lopez teaches that these are known and classic methods for preparing a protein-containing sample for analysis by mass spectrometry.
Applicant’s claimed invention does not present non-obvious limitations or combination of limitations, but rather relies on well-known concepts and techniques of protein analysis that comprise immobilizing the protein on magnetic beads that are provided in an oscillating magnetic field for stirring with known reagents for contact and reaction in an analysis, such as stirring with a denaturing solution for denaturing proteins as a pretreatment for analysis by mass spectrometry, and also contacting with other well-known reagents for enzymatically digesting and processing the enzymes before the analysis by mass spectrometry.
As to claim 2, Zhang teaches that the characterization comprises analyzing the plurality of peptides comprising LC-MS/MS [see paras. 0011, 0023, 0025-0026.]
As to claim 17, Zhang teaches that the first magnetic beads do not comprise an immobilized endopeptidase enzyme. [See para. 0022, 0024, 0049, disclosing blank beads.]
As to claim 18, the changing magnetic field taught by Imagawa is an electromagnetic mixer. See paragraphs 0019-0021 disclosing electromagnets.
As to claims 19 and 39, Imagawa teachings providing an oscillating magnetic field as discussed above. AC driven oscillating magnetic field is well-known in the art.
Moreover, Imagawa discloses the following.
The size of the “motor” is limited as long as it can generate power by using electricity or the like as an energy source and can rotate a member such as a magnet holder that holds the magnet with the power. Not something. For example, a small motor (manufactured by Mabuchi Co., Ltd .: rotation speed 7800 rpm, 130 rotations / second) or the like can be used. Of course, this motor can also be equipped with more than one motor to get more power. Para. 0022.
As exemplified in FIG. 1, the reaction promoting device for the reaction promoting method includes a magnet (1), a magnet holder (2) on which the magnet (1) is disposed and held, and a magnet holder. A motor (3) serving as a power source for rotating (2) is provided. In the example of FIG. 1, a pair of magnets (1) are disposed and held on a magnet holder (2) to face each other in order to apply rotational vibration to the magnetic fine particles in the sample. A vial (5) containing a magnetic particle-containing solution (4) as a sample solution is placed between the pair of magnets (1). Next, power is generated by operating the motor (3) with electricity or the like, and the generated power is transmitted to the magnet holder (2). Then, by rotating (arrow) the magnet (1) disposed and held on the magnet holder (2) around the vial (5), the magnetic fine particles are rotationally vibrated in the sample solution. The reaction between the characteristic component to be analyzed and the active component immobilized on the magnetic fine particles is promoted. Para. 0023.
As to claim 37, the oscillation disclosed by Imagawa stirs the magnetic particles (paras. 0010 and 0013). Examiner notes that such oscillation and stirring in effects disaggregates the magnetic beads.
As to claim 38, the oscillating magnetic field disclosed by Imagawa inherently causes three-dimensional mixing of the aqueous solution by the magnetic beads.
As to claim 40, the oscillating magnetic field causes the beads to act as micro-stirring bars to stir the aqueous solution [see para. 0013, disclosing stirring using magnetic beads and an oscillating magnetic field].
Claims 7-16 is/are rejected under 35 U.S.C. 103 as being unpatentable over CN 103675115 (hereinafter “Zhang”) in view of JP 2004309189 (hereinafter “Imagawa”), and US 20190086306 (hereinafter “Lopez”), as applied to claims 1 and 3 above, and further in view of US 20070128729 (hereinafter “Puente”).
As to claims 7 and 8, Zhang, Imagawa, and Lopez, discussed above, are silent as to an endopeptidase enzyme being immobilized.
However, Lopez does teach a method for preparing a protein-containing sample for analysis by mass spectrometry which includes introducing an immobilized proteolytic enzyme, a reducing agent and an alkylating agent (see for example, paras. 0006-0008).
Moreover, Puente show that endopeptidase such as trypsin is used for enzyme pretreatment [digesting] for protein analysis by mass spectrometry (see claim 1 in Puente), and thus it would have been obvious to one skilled in the art to utilize endopeptidase, or specifically trypsin, in the Zhang-Imagawa-Lopez invention in order to analyte protein by mass spectrometry.
As to claim 9, Puente does not requires that the endopeptidase enzyme be a free endopeptidase enzyme or an immobilized endopeptidase enzyme, and therefore it would have been obvious that it could be either. Moreover, Lopez discloses an immobilized proteolytic enzyme (see for example para. 006-008.)
As to claim 10, multiple beads are taught by Zhang (see discussion of Zhang above).
As to claim 11, Imagawa teaches that the beads [and therefore the claimed first magnetic beads and the second magnetic beads independently comprise ferromagnetic or superparamagnetic particles (see Imagawa, para. 0015).
As to claim 12, while Zhang is silent as to the first magnetic beads and the second magnetic beads independently have a high magnetic response having a Bmax in a range of from about 20 emu/g to about 250 emu/g, 40 emu/g to 200 emu/g, 50 emu/g to 150 emu/g. or 80 emu/g to 100 emu/g, such a magnetic response falls within a workable or optimum range, and thus its discovery would have required ordinary skills in the art, given that Zhang, Imagawa, Lopez, and Puente teaches the general conditions of the claims, as discussed above.
As to claim 13, while Zhang is silent as to the first magnetic beads and/or the second magnetic beads independently have a high surface area of> 5 m2/g, such range falls within a workable or optimum range, and thus its discovery would have required ordinary skills in the art, given that Zhang, Imagawa, Lopez, and Puente teaches the general conditions of the claims, as discussed above.
As to claim 14, the enzyme taught by Lopez and Puente is equivalent to the claimed coating.
As to claim 15, enzyme taught by Lopez and Puente is equivalent to the claimed functional group-coated surface.
As to claim 16, the protein or antibody or any part of its chemical composition taught by Zhang, Lopez, or Puente is equivalent to the claimed bioaffinity adsorbent.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over CN 103675115 (hereinafter “Zhang”) in view of JP 2004309189 (hereinafter “Imagawa”), and further in view of US 20190086306 (hereinafter “Lopez”), as applied to claim 3 above, and further in view of WO 2015055825 (hereinafter “Poglitsch”).
While Zhang, Imagawa, and Lopez, discussed above, are silent as to the chaotropic agent being ethanol, Poglitsch discloses that this is a known chaotropic agent (see para. 0104), and thus its use requires ordinary skills in the art as a known chaotropic agent, as shown by Poglitsch, with reasonable expectation of success as a substitute chaotropic agent in the modified Zhang invention.
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over CN 103675115 (hereinafter “Zhang”) in view of JP 2004309189 (hereinafter “Imagawa”), and further in view of US 20190086306 (hereinafter “Lopez”), as applied to claim 3 above, and further in view of US 20150126402 (hereinafter “Shabb”).
While Zhang, Imagawa, and Lopez (discussed above) is silent as to iodoacetic acid being the alkylating agent, Shabb discloses that this is a known alkylating agent (para. 0289), and thus its use requires ordinary skills in the art as a known alkylating agent, as shown by Shabb, with reasonable expectation of success as a substitute alkylating agent in the modified Zhang invention.
Response to Arguments
Applicant has amended the claims and argues that Neely fails to teach using magnetic beads to stir or use of an oscillating magnetic field.
Examiner has cited a new reference (Imagawa) to teach an oscillating magnetic field to obtain a stirring effect (see para. 0013 as well as throughout the reference, specifically mentioning stirring). Therefore, Applicant’s remarks regarding Neely is moot.
Applicant also requests a human translation of Zhang and asserts that the Office-supplied machine translation does not accurately represent the contents of Zhang. Applicant also argues that Zhang does not teach use of an oscillating magnetic field.
Examiner has not asserted that Zhang teaches an oscillating magnetic field, and therefore a secondary reference was cited (Imagawa in the present Office action). Examiner has placed a request for a human translation of Zhang, which will be submitted to Applicant once provided to Examiner.
Conclusion
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/Ann Montgomery/Primary Examiner, Art Unit 1678
