Ex Parte MayerDownload PDFBoard of Patent Appeals and InterferencesSep 23, 200910426169 (B.P.A.I. Sep. 23, 2009) Copy Citation UNITED STATES PATENT AND TRADEMARK OFFICE __________ BEFORE THE BOARD OF PATENT APPEALS AND INTERFERENCES __________ Ex parte BRENT A. MAYER __________ Appeal 2009-005415 Application 10/426,169 Technology Center 1600 __________ Decided: September 23, 2009 __________ Before TONI R. SCHEINER, ERIC GRIMES, and STEPHEN WALSH, Administrative Patent Judges. WALSH, Administrative Patent Judge. DECISION ON APPEAL This is an appeal under 35 U.S.C. § 134(a) involving claims to methods for detecting an analyte in an enzyme-linked immunosorbent assay and to enzyme immunoassays. The Patent Examiner rejected the claims as obvious. We have jurisdiction under 35 U.S.C. § 6(b). We affirm. Appeal 2009-005415 Application 10/426,169 STATEMENT OF THE CASE The invention relates to enzyme-linked immunosorbent assays, aka ELISAs, which are said to be “the most commonly used immunoassays for a wide variety of applications in diagnostics, research, food testing, process quality assurance and quality control, and environmental testing.” (Spec. 2:5-9.) In the prior art, ELISA could be done with a colorless or nearly colorless chromogenic substrate, and the presence (or absence) of a particular analyte in the sample being tested was detected or measured to the extent the chromogenic substrate developed color. When the ELISA used peroxidase as the enzyme, the ELISA might include an acid stop following oxidation of the chromogenic substrate. (Id. at 3:12-15.) According to the Specification, the typically colorless nature of substrates and samples led to two problems in prior art assays. First, “it is difficult to determine immediately by visual examination whether the chromogenic substrate had been added to a particular tube or well or not.” (Id. at pp. 2-3.) The second problem was “determining whether a negative reaction is negative because the sample for the reaction did not contain the analyte being tested for or is negative because one or more reagents necessary for the reaction had been omitted, for example the colorless chromogenic substrate.” (Id. at 3:6-12.) Appellant’s method adds a colorless pH indicator dye at the start of the assay. When a stop solution is later added, the stop solution causes the pH indicator dye to turn color, confirming that a substrate had been added. According to the Specification: The present invention provides a solution to the problem of whether a negative reaction in enzyme-linked assays such as ELISAs (direct, indirect, and competitive), immunoassays 2 Appeal 2009-005415 Application 10/426,169 (direct, indirect, and competitive), hybridizations, or the like, is negative because the test sample did not contain the analyte being tested for or is negative because one or more reagents necessary for the reaction had been omitted by including in a ready-to-use composition for enzyme-linked assays a pH indicator dye which produces a detectable color or absorbance when a stop solution is added to the composition in the reaction vessel. (Id. at 24:10-21.) Claims 1, 3, 5, 8-15, 17, 22, 24, 26 and 29-38, which are all the pending claims, are on appeal. Claim 1 is representative and reads as follows: 1. A method for detecting the presence or absence of an analyte in a sample in a peroxidase-based enzyme-linked immunosorbent assay (ELISA) and determining whether a false negative reaction has occurred, which comprises: (a) providing a composition comprising a mixture of a chromogenic substrate which is oxidizable by peroxide in a reaction for the ELISA catalyzed by the peroxidase of the ELISA to form a first color with an absorbance at a first wavelength, a pH indicator dye which is colorless or has a color essentially not detectable by the eye when applied to the one or more reaction vessels and forms a second color which is detectable by eye and has an absorbance at a second wavelength when a stop solution is added to the composition in one or more reaction vessels, and a peroxide; (b) adding an aliquot of the composition to each of the one or more reaction vessels containing the sample for the ELISA to form a reaction mixture; (c) incubating the reaction mixture for a time sufficient to oxidize the chromogenic substrate to the first color; (d) adding the stop solution to the reaction mixture to stop the reaction and to generate the second color; (e) measuring spectrophotometrically absorbances at the first wavelength for each of the reaction vessels; 3 Appeal 2009-005415 Application 10/426,169 (f) detecting the second color by eye, wherein the second color indicates that the reaction mixture contains the composition and the stop solution; (g) determining from step (f) whether the false negative reaction has occurred in each of the reaction vessels by the absence of an absorbance at the second wavelength or absence of the second color; and (h) detecting the presence or absence of the analyte by the absorbance at the first wavelength as measured in step (e), in each of the reaction vessels where a false negative reaction has been determined not to have occurred in step (g), wherein the absorbance at the first wavelength indicates the presence or absence of the analyte. The Examiner rejected claims 1, 3, 5, 8-15, 17, 22, 24, 26 and 29-38 under 35 U.S.C. § 103(a) as unpatentable over the combined teachings of Lihme1 and Duncan.2 Appellant states that all the rejected claims “are grouped and argued with claim 1.” (App. Br. 16.) Accordingly, claims 3, 5, 8-15, 17, 22, 24, 26 and 29-38 stand or fall with claim 1. 37 C.F.R. § 41.37(c)(1)(vii). OBVIOUSNESS The Issue The Examiner’s position is that Lihme taught an ELISA method in which a visible dye was included in a ready-to-use substrate composition, ensuring that the addition of substrate to a reaction mixture was readily observable. (Ans. 3.) When a stop solution was later added, the dye would turn colorless. (Id. at 4.) The Examiner noted that Lihme listed suitable 1 U.S. Patent No. 6,221,624 B1, issued to Allan Otto Fog Lihme et al., Apr. 24, 2001. 2 WO 93/25905, by Richard Duncan et al., published Dec. 23, 1993. 4 Appeal 2009-005415 Application 10/426,169 dyes, including m-cresol purple, a dye that Appellant’s method uses (see Appellant’s claim 5). (Id.) The Examiner found that the difference between Lihme’s method and Appellant’s method is that Lihme employed different reactions conditions: Specifically, in Lihme et al. the ready-to-use substrate compositions are provided at a pH at which the pH indicator dye will be initially colored, rather than initially colorless (see especially Example 1). Addition of the stop solution at the end of the assay then produces a colorless (or differently colored) form of the dye. (Id. at 4-5.) The Examiner found that “[l]ike Lihme et al., Duncan et al. also address the problem of false negative results arising in immunassays due to the inadvertent omission of necessary reagents.” (Id. at 5.) Duncan taught “adding sample monitor reagents, which may include pH indicator dyes, to the sample. The sample monitor reagent is then subsequently detected (e.g., by detecting a color change) in order to confirm that the sample has been correctly added.” (Id. at 5, citations omitted.) Duncan taught the detection of the sample monitor reagent could be done as a “preliminary step or after the assay proper.” (Id. at 6, citations omitted.) Thus, “the results of the control detection step and the results of the assay proper may be compared at the end of the assay.” (Id.) Citing Duncan’s disclosure at pp. 18-19, the Examiner found that Duncan was cognizant of the possibility that the sample monitor signal and the chromogenic substrate signal could interfere, and provided guidance as to how to avoid signal interference. The Examiner summarized Duncan’s disclosure as follows: The teachings of Duncan et al. establish that it was known to use pH indicator dyes in immunoassays in order to 5 Appeal 2009-005415 Application 10/426,169 rule out false negative reactions, and further that the presence of such dyes can be detected either visually or spectrophotometrically at different points during the assaying, including at the end of the assay when the analyte is measured. (Id.) Appellant contends that there is no prima facie case of obviousness and presents “two specific issues for consideration on appeal: (1) Whether the applied references teach away from the recited methods; and, (2) Whether the obviousness rejection is sufficiently supported by a reason prompting the proposed combination of references.” (App. Br. 16.) To support the teaching away argument, Appellant contends that “[t]he intended purpose of Lihme is not simply to detect possible false negatives . . . . Rather, the intended purpose is to improve the substrate pipetting step of an assay.” (Id. at 19, emphasis in original.) That is, Lihme “solved [the colorless substrate problem] by adding a visible dye that permits a verification of substrate addition during the pipetting stage of the assay.” (Id.) Appellant contends that “the visibility of the dye in the substrate solution . . . is an essential feature of [Lihme’s] disclosed dye.” (Id.) Appellant acknowledges that Duncan taught that the order of colorimetric sample detection can be varied, but argues that Lihme “requires” detection at the pipetting stage, prior to addition of a stop solution. (Id. at 20.) In Appellant’s view, “the combined references simply teach the use of a soluble dye that is initially colored and visible,” and “lead 6 Appeal 2009-005415 Application 10/426,169 the skilled artisan ‘in a direction divergent from the path that was taken by the applicant’ and teach away from the claimed methods. In re Kahn, 441 F.3d at 999.” (Id.) Further, “the proposed modification impermissibly renders Lihme unsatisfactory for its intended purpose.” (Id.) For the second issue, Appellant cites to the Examiner’s finding that one of ordinary skill in the art would have been motivated to modify Lihme’s method to allow for detecting the dye at the end of, rather than during, the assay. (Id. at 20.) Appellant points to the portion of Duncan’s disclosure that purportedly taught “visible detection of a sample prior to the actual assay.” (Id. at 20-21.) Asserting that both references taught “that such visible detection should be performed prior to the assay,” Appellant argues that the Examiner’s reasoning does not follow from the relied-upon sections and fails to provide a reason to combine the references in the manner proposed by the rejection. (Id. at 21.) The issues on appeal are: did Lihme and Duncan teach away from the claimed methods; and did the Examiner provide a sufficient reason prompting the combination of teachings from Lihme and Duncan? Findings of Fact 1. Lihme’s patent is entitled “Pre-Stained 3,3’,5,5’-tetramethylbenzidine Substrates For The Detection Of Enzyme Activity.” 2. Lihme described a problem with ELISA assays at the time: pipettting of the thus colorless substrate into the enzyme immunoassay reaction vessel, which often is a transparent tube or a transparent ELISA tray, is associated with the risk of incorrect dosage in that it may 7 Appeal 2009-005415 Application 10/426,169 be difficult to determine immediately by visual examination whether the substrate has been added to a vessel (e.g. a well) or not. (Col. 2, ll. 1-7.) 3. Lihme “provide[d] a solution to the problems associated with applying an otherwise colourless substrate to a reaction vessel . . . by including a dye in ready-to-use substrates.” (Col. 2, ll. 55-59.) 4. Lihme taught: “[i]n particular, the present invention provides a storage-stable pre-stained 3,3’,5,5’-tetramethylbenzidine (TMB) substrate comprising: (a) a water-based TMB substrate composition which includes a peroxide and (b) a dye which is soluble therein.” (Col. 3, ll. 44-48.) 5. Lihme taught dyes with suitable properties including “m-Cresol Purple.” (Col. 5, ll. 45-52.) 6. Duncan provided a method “for determining false negative results in an immunoassay . . . by determining the presence of [a] sample in an immunoassay vessel . . . .” (Abstract.) 7. Duncan’s method “comprise[d] using a sample monitor reagent or reagent system that is capable of producing a signal when sample is present . . . .” (Id.) 8. In Duncan’s method, “[a] negative signal generated by the sample monitor reagent or reagent [system] denotes a false negative result in the immunoassay itself.” (Id.) 9. According to Duncan, [t]he present invention is based on our appreciation that it is important that users should be able to ascertain readily whether sample has, in fact, been added to each 8 Appeal 2009-005415 Application 10/426,169 immunoassay vessel to which it should be added because the absence of sample is one cause of false negative results. (Duncan at 4:19-23.) 10. Duncan taught a method of mixing sample with sample monitor reagent, then adding the mixture to an assay vessel, and then detecting the sample monitor signal. (Id. at 5:4-20.) 11. Although the sample monitor step was “generally carried out as a preliminary step,” “[i]f desired, however, the order of sample monitor step and the assay proper may be varied.” (Id. at 6:11-16.) 12. Duncan taught using a color change as the sample monitor signal. (Id. at 10:37-38.) 13. Duncan instructed: “the choice of [sample monitor] reagent should preferably take into account the pH of the particular assay system in which it will be used.” (Id. at 12:13-19.) 14. The Examiner found that “the principle of detecting changes in color of a pH indicator dye as an indication of the presence of the dye was well known in the art.” (Ans. 7.) 15. The Examiner found that one of ordinary skill in the art at the time of the invention would have been motivated to modify Lihme’s method by providing an initially colorless dye and detecting its presence at the end of the assay “in order to achieve the same purpose, namely to detect the presence of the dye and thus the presence of the substrate composition in the assay so as to rule out false negatives.” (Id.) 16. The Examiner found a further source of motivation for later detection was the advantage of “allowing the assay to be carried out from start 9 Appeal 2009-005415 Application 10/426,169 to finish without stopping to visually inspect the reaction (as taught by Duncan et al.).” (Id.) 17. The Examiner found that “[t]he convenience of detecting both the analyte (via TMB absorbance) and the control pH indicator dye at the end of the assay, rather than having to interrupt the assay to detect the control dye, provides motivation to modify the teachings of Lihme et al.” (Id. at 7-8.) 18. The Examiner found that motivation to combine the references “arises from the fact that they both relate to the same problem to be solved, namely that of false negative reactions.” (Id. at 8.) Principles of Law A prior art reference is said to teach away from an applicant’s invention “when a person of ordinary skill, upon reading the reference, would be discouraged from following the path set out in the reference, or would be led in a direction divergent from the path that was taken by the applicant.” In re Gurley, 27 F.3d 551, 553 (Fed. Cir. 1994). “The prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed.” In re Fulton, 391 F.3d 1195, 1201 (Fed. Cir. 2004). A rejection for obviousness must include “articulated reasoning with some rational underpinning to support the legal conclusion.” KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 418 (2007), quoting In re Kahn, 441 F.3d 977, 988 (Fed. Cir. 2006). The proper question to ask is whether a person of ordinary skill in the art, facing the wide range of needs created by 10 Appeal 2009-005415 Application 10/426,169 developments in the field of endeavor, would have seen a benefit to combining the prior art teachings. KSR, 550 U.S. at 424; e.g., Cross Med. Prods., Inc., v. Medtronic Sofamor Danek, Inc., 424 F.3d 1293, 1322 (Fed. Cir. 2005) (“The problem was within the general knowledge of those of ordinary skill in the art, and thus provided sufficient motivation to navigate the prior art in the spinal implant field in search of a teaching on how one might modify the '602 device away from a bottom-tightening assembly.”); Princeton Biochemicals, Inc. v. Beckman Coulter, Inc., 411 F.3d 1332, 1339 (Fed. Cir. 2005) (“[T]he nature of the problem also supplies a motivation to make this combination of closely related prior art elements.”). A reference must be read, not in isolation, but for what it fairly teaches in combination with the prior art as a whole. In re Merck & Co., Inc., 800 F.2d 1091, 1097 (Fed. Cir. 1986). “[I]f a technique has been used to improve one device, and a person of ordinary skill in the art would recognize that it would improve similar devices in the same way, using the technique is obvious unless its actual application is beyond his or her skill.” KSR, 550 U.S. at 417. Analysis Step (a) in Appellant’s claim 1 provides a composition comprising a chromogenic substrate, a colorless pH indicator dye, and a peroxide. Lihme taught an assay method that provided a composition comprising the same three ingredients. (FF4.) Appellant claims using m-cresol purple as an indicator dye. See claim 5. Lihme taught using m-cresol purple as the indicator dye. (FF5.) We agree with the Examiner’s finding that 11 Appeal 2009-005415 Application 10/426,169 Appellant’s method starts with a composition comprising the same ingredients as Lihme’s method used. We also agree with the Examiner’s finding concerning the difference between the claimed method and Lihme’s method. The Examiner found that the difference is that Lihme taught starting the assay at a pH where m-cresol purple has color, but Appellant’s method begins the assay at a pH where m- cresol purple is colorless. (Ans. 4-5.) Appellant does not dispute this finding, but does dispute the Examiner’s reliance on Duncan. The Examiner found that Duncan addressed the problem of false negative ELISA results associated with sample errors. To address the possibility that a worker could forget to include the sample, Duncan added a “sample monitor reagent” to the sample. (FF7.) The sample monitor reagent was a dye that could undergo a color change. (FF12.) Duncan taught that the sample monitor reagent could be observed at the end of the assay. (FF11.) Duncan taught that the choice of sample monitor reagent should take into account the pH of the particular assay system being used (FF13), and the Examiner found that Duncan evidences that “it was known to use pH indicator dyes in immunoassays in order to rule out false negative reactions, and further that the presence of such dyes can be detected either visually or spectrophotometrically at different points during the assaying, including at the end of the assay when the analyte is measured.” (Ans. 6.) We agree that the evidence supports this finding and Appellant does not dispute the finding. The Examiner found several motivational suggestions in the prior art, and concluded that the claimed methods would have been obvious. (FF15- 18.) The evidence supports the Examiner’s findings with regard to 12 Appeal 2009-005415 Application 10/426,169 motivation. Appellant contends however, that Lihme and Duncan “teach away” from the claimed methods, and that the Examiner failed to provide a “sufficient” reason for combining their teachings. We do not agree that Appellant has established that the references teach away from the claimed methods. While Lihme used a pH indicator dye that had color at the beginning pH of the assay, Lihme is silent on whether the same dye could be used in an assay that starts at a pH where the dye is colorless, but ends at a pH where the dye has a color, and its color is then detected, as expressly taught by Duncan. The mere fact that Lihme was silent about the method suggested with Duncan does not mean that the two references, taken together, lead away from the method claimed. We must weigh the teachings of the references in light of each other, not in isolation. See Merck, 800 F.2d at 1097. Appellant has not disputed the Examiner’s finding of the level of skill and knowledge in the art (FF14), and we agree with the Examiner that a person of ordinary skill in the art would have understood Duncan’s disclosure of alternative times for observing the sample monitoring reagent to fairly suggest the claimed method. See Fulton, 391 F.3d at 1201. We reject Appellant’s contention that the Examiner failed to state a sufficient reason for combining the prior art teachings. Both references addressed known problems in the ELISA art. The evidence shows that false negatives due to substrate and sample errors were part of the general knowledge in the art. Lihme and Duncan specifically addressed those known problems. General knowledge in the art and the nature of the problem addressed are both proper sources for reasons to combine prior art teachings. E.g., Cross Med. Prods., 424 F.3d 1293 at 1322; Princeton 13 Appeal 2009-005415 Application 10/426,169 Biochemicals, 411 F.3d at 1339. The Examiner’s analysis provided multiple reasons for combining the teachings of the references (FF15-18), and explained why there would have been a reasonable expectation of success for combining the teachings (Ans. 8-9.) We find that (1) the Examiner’s statements had a rational underpinning based on identified facts, see KSR, 550 U.S. at 418; (2) a person of ordinary skill in the art at the time would have seen the benefits to combining the prior art teachings that the Examiner identified, see KSR, 550 U.S. at 424; and (3) a person of ordinary skill in the art at the time would have expected the Duncan method to be useful in the similar Lihme method, see KSR, 550 U.S. at 417. CONCLUSIONS OF LAW Lihme and Duncan did not teach away from the claimed methods. The Examiner provided a sufficient reason prompting the combination of teachings from Lihme and Duncan. SUMMARY We affirm the rejection of claims 1, 3, 5, 8-15, 17, 22, 24, 26 and 29- 38 under 35 U.S.C. § 103(a) as unpatentable over the combined teachings of Lihme and Duncan. No time period for taking any subsequent action in connection with this appeal may be extended under 37 C.F.R. § 1.136(a). AFFIRMED 14 Appeal 2009-005415 Application 10/426,169 cdc IAN C. McLEOD, P.C. 2190 COMMONS PARKWAY OKEMOS MI 48864 15 Copy with citationCopy as parenthetical citation