Ex Parte Mayer

Board of Patent Appeals and Interferences

Sep 23, 2009

10426169 (B.P.A.I. Sep. 23, 2009)

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