U.S. patent application number 10/480314 was filed with the patent office on 2004-10-28 for total protein detection methods and devices.
Invention is credited to Farr, Jennifer, Orn, Alexander H., Profitt, James A..
Application Number | 20040214339 10/480314 |
Document ID | / |
Family ID | 23157725 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040214339 |
Kind Code |
A1 |
Profitt, James A. ; et
al. |
October 28, 2004 |
TOTAL PROTEIN DETECTION METHODS AND DEVICES
Abstract
An assay for the determination of protein in an aqueous test
fluid which combines the test fluid with a buffer and a dye. The
buffer is selected from citrulline, malonic acid, cyanoacetic acid,
citraconic acid, methyl phosphonic acid, sarcosine, saccharin, or
combinations thereof. The buffer is added in sufficient quantity to
maintain the pH of the assay including the test fluid at a selected
target pH range within a range of from about 2.0 to about 3.0. The
dye has a pKa which enables it to operate as a protein indicator at
the target pH range. The dye also has affinity for protein such
that it will provide a detectable response in the presence of
greater than about 15 mg/dL protein to thereby render the assay
suitable for the detection of total protein in the test fluid. The
buffer and dye may be absorbed in a test strip of absorbent
material.
Inventors: |
Profitt, James A.; (Goshen,
IN) ; Orn, Alexander H.; (Nappanee, IN) ;
Farr, Jennifer; (Albion, IN) |
Correspondence
Address: |
Elizabeth A Levy
Bayer Healthcare
63 North Street
Medfield
MA
02052
US
|
Family ID: |
23157725 |
Appl. No.: |
10/480314 |
Filed: |
December 11, 2003 |
PCT Filed: |
June 21, 2002 |
PCT NO: |
PCT/IB02/02321 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60300104 |
Jun 25, 2001 |
|
|
|
Current U.S.
Class: |
436/86 ;
422/424 |
Current CPC
Class: |
G01N 33/543 20130101;
G01N 33/6839 20130101 |
Class at
Publication: |
436/086 ;
422/056 |
International
Class: |
G01N 021/00 |
Claims
1. In an assay for the determination of protein in an aqueous test
fluid which comprises combining the test fluid with (a) a buffer
selected from the group consisting of citrulline, cyanoacetic acid,
methyl phosphonic acid, saccharin, or combinations thereof, the
buffer is added in sufficient quantity to maintain the pH of the
assay including the test fluid at a selected target pH range within
a range of from about 2.0 to about 3.0 and (b) a dye having a pKa
which enables it to operate as a protein indicator at the target pH
range and whose affinity for protein is such that it will provide a
detectable response in the presence of greater than about 15 mg/dL
protein to thereby render the assay suitable for the detection of
total protein in the test fluid.
2. The assay of claim 1, wherein the buffer is selected from the
group consisting of citraconic acid, methyl phosphonic acid,
saccharin, or combinations thereof.
3. The assay of claim 1, wherein the buffer is added in a
sufficient quantity to maintain the pH of the assay including the
test fluid within a range of from about 2.4 to about 3.0.
4. The assay of claim 1, wherein the dye is a substituted
phenolsulfonephthalein, pyrogallol red or combinations thereof.
5. The assay of claim 4, wherein the dye is a substituted
phenolsulfonephthalein, the substituted phenolsufonephthalein is
octasubstituted with amino, amido, aromatic, alkyl, hydroxyl, thio,
carboxylic, alkoxy, ketoxy, acetyl, halogen, nitro, cyano, or
combinations thereof.
6. The assay of claim 4, wherein the dye is a substituted
phenolsulfonephthalein, the substituted phenolsulfonephthalein is
substituted at the 5', 5 " positions with nitro and at the 3', 3",
3, 4, 5 and 6 positions with chloride, bromide or iodide.
7. The assay of claim 4, wherein the dye is a substituted
phenolsulfonephthalein, the substituted phenolsulfonephthalein is
5,5"-Dinitro-3', 3",3,4,5,6-hexabromophenolsulfonephthalein or
5'-Nitro-5"-Iodo-3',
3",3,4,5,6,-hexabromophenolsulfonephthalein.
8. The assay of claim 1, wherein the dye is pyrogallol red and
further includes a molybdate or tungstate salt.
9. The assay of claim 1 wherein the buffer and dye are absorbed in
a test strip of absorbent material.
10. The assay of claim 1, wherein the detectable response provides
good resolution at protein concentrations of from about 15 to about
300 mg/dL.
11. The assay of claim 1, wherein the dye operates as a color
indicator in the presence of protein at a pH within the range of
from about 2.0 to about 3.0.
12. The assay of claim 11, wherein the dye operates as a color
indicator in the presence of protein at a pH within the range of
from about 2.4 to about 3.0.
13. The assay of claim 1, wherein the buffer maintains the pH of
the assay including the test fluid at a target pH plus or minus
about 0.2 pH units.
14. In an assay for the determination of protein in an aqueous test
fluid which comprises combining the test fluid with (a) a buffer
selected from the group consisting of citrulline, cyanoacetic acid,
methyl phosphonic acid, saccharin, or combinations thereof, the
buffer is added in sufficient quantity to maintain the pH of the
assay including the test fluid within about +/-0.2 pH units of a
selected target pH range within a range of from about 2.0 to about
3.0 and (b) a dye having a pKa which enables it to operate as a
color indicator in the presence of protein at the target pH range
and whose affinity for protein is such that it will provide a
detectable response in the presence of greater than about 2 to
about 500 mg/dL protein to thereby render the assay suitable for
the detection of total protein in the test fluid.
15. A dry device for use in an assay for determining protein levels
in a fluid test sample which comprises an absorbent material having
absorbed therein (a) a buffer selected from the group consisting of
citrulline, cyanoacetic acid, methyl phosphonic acid, saccharin, or
combinations thereof, the buffer is added in sufficient quantity to
maintain the pH of the assay including the test fluid at a selected
target pH range within a range of from about 2.0 to about 3.0 and
(b) a dye having a pKa which enables it to operate as a protein
indicator at the target pH range and whose affinity for protein is
such that it will provide a detectable response in the presence of
greater than 15 mg/dL protein upon contact between the device and
the fluid test sample to thereby render the device suitable for the
detection of total protein in the fluid test sample.
16. The dry device of claim 15, wherein the buffer is selected from
the group consisting of cyanoacetic acid, methyl phosphonic acid,
saccharin, or combinations thereof.
17. The dry device of claim 15, wherein the buffer is added in a
sufficient quantity to maintain the pH of the assay including the
test fluid within a range of from about 2.4 to about 3.0.
18. The dry device of claim 15, wherein the dye is a substituted
phenolsulfonephthalein, pyrogallol red or combinations thereof.
19. The dry device of claim 18, wherein the dye is a substituted
phenolsulfonephthalein, the substituted phenolsulfonephthalein is
octasubstituted with amino, amido, aromatic, alkyl, hydroxyl, thio,
carboxylic, alkoxy, ketoxy, acetyl, halogen, nitro, cyano, or
combinations thereof.
20. The dry device of claim 18, wherein the dye is a substituted
phenolsulfonephthalein, the substituted phenolsulfonephthalein is
substituted at the 5', 5" positions with nitro and at the 3', 3",
3, 4, 5 and 6 positions with chloride, bromide or iodide.
21. The dry device of claim 18, wherein the dye is a substituted
phenolsulfonephthalein, the substituted phenolsulfonephthalein is
5,5"-Dinitro-3',3",3,4,5,6-hexabromophenolsulfonephthalein or
5'-Nitro-5"-Iodo-3',
3",3,4,5,6,-hexabromophenolsulfonephthalein.
22. The dry device of claim 15, wherein the dye is pyrogallol red
and further includes a molybdate or tungstate salt.
23. The dry device of claim 15, wherein the detectable response
provides good resolution at protein concentrations of from about 15
to about 300 mg/dL.
24. The dry device of claim 15, wherein the dye operates as a color
indicator in the presence of protein at a pH within the range of
from about 2.0 to about 3.0.
25. The dry device of claim 24, wherein the dye operates as a color
indicator in the presence of protein at a pH within the range of
from about 2.4 to about 3.0.
26. The dry device of claim 15, wherein the buffer maintains the pH
of the assay including the test fluid at a target pH plus or minus
about 0.2 pH units.
27. A dry device for use in an assay for determining protein levels
in a fluid test sample which comprises an absorbent material having
absorbed therein (a) a buffer selected from the group consisting of
citrulline, cyanoacetic acid, methyl phosphonic acid, saccharin, or
combinations thereof, the buffer is added in sufficient quantity to
maintain the pH of the assay including the test fluid within about
+/-0.2 pH units of a selected target pH range within a range of
from about 2.0 to about 3.0 and (b) a dye having a pKa which
enables it to operate as a color indicator in the presence of
protein at the selected target pH range and whose affinity for
protein is such that it will provide a detectable response in the
presence of from about 2 to about 500 mg/dL protein upon contact
between the device and the fluid test sample to thereby render the
device suitable for the detection of total protein in the fluid
test sample.
28. In an assay for the determination of protein in an aqueous test
fluid which comprises combining the test fluid with (a) as a buffer
citrulline, the buffer is added in sufficient quantity to maintain
the pH of the assay including the test fluid at a selected pH range
within a range of from about 2.0 to about 3.0 and (b) a dye having
a pKa which enables it to operate as a protein indicator at the
target pH range and whose affinity for protein is such that it will
provide a detectable response in the presence of greater than about
15 mg/dL protein to thereby render the assay suitable for the
detection of total protein in the test fluid.
29. Cancelled.
30. Cancelled.
31. The assay of claim 28, wherein the dye is a substituted
phenolsulfonephthalein, pyrogallol red or combinations thereof.
32. The method assay of claim 28, wherein the dye operates as a
color indicator in the presence of protein at a pH within the range
of from about 2.0 to about 3.0.
33. The assay of claim 32, wherein the dye operates as a color
indicator in the presence of protein at a pH within the range of
from about 2.4 to about 3.0.
34. A dry device for use in an assay determining protein levels in
a fluid test sample which comprises an absorbent material having
absorbed therein (a) as a buffer citrulline, the buffer is added in
sufficient quantity to maintain the pH of the assay including the
test fluid at a selected target pH range within a range of from
about 2.0 to about 3.0 and (b) a dye having a pKa which enables it
to operate as a protein indicator at the target pH range and whose
affinity for protein is such that it will provide a detectable
response in the presence of greater than 15 mg/dL protein upon
contact between the device and the fluid test sample to thereby
render the device suitable for the detection of total protein in
the fluid test sample.
35. Cancelled.
36. Cancelled.
37. The dry device of claim 34, wherein the dye is a substituted
phenolsulfonephthalein, pyrogallol red or combinations thereof.
38. The dry device of claim 34, wherein the dye operates as a color
indicator at a pH within the range of from about 2.0 to about 3.0
in the presence of protein.
39. The dry device of claim 38, wherein the dye operates as a color
indicator at a pH within the range of from about 2.4 to about 3.0
in the presence of protein.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to methods and
devices of detecting proteins. Specifically, the invention is
directed to methods and devices for detecting proteins at lower
pH's using various buffers.
BACKGROUND OF THE INVENTION
[0002] Methods for the detection of urinary proteins are often more
sensitive to albumin than to other urinary proteins. It is
important, however, to detect proteins other than albumin,
especially in the case of Bence-Jones proteinuria. The detection of
proteins should cover a wide range of protein concentrations since
the decision levels and recommended actions to be taken by
clinicians will vary depending on the concentration of protein
detected in a patient's urine. For example, persistent proteinuria
greater than 50 mg/dL represents strong evidence of renal disease
whereas a protein level of greater than 300 mg/dL is consistent
with a diagnosis of nephrotic syndrome. A concentration of protein
in urine of greater than 800 mg/dL suggests massive protein loss
and warrants a renal biopsy and/or steroid therapy. Accordingly, it
is apparent that a test for protein in urine should be effective
over a wide range of protein values.
[0003] Various methods for the determination of protein in aqueous
fluid have been reported in the literature. These methods include
the Kjeldahl method, biuret method, Lowery method, dyestuff
combination method, UV method and fluorometric method. In general,
proteins react with various substance including dyes such as
bromphenol blue, coomassie brilliant blue and eosine, as well as
metal ions such as silver (I), copper (II), zinc (II) and lead
(II).
[0004] Urine contains amounts of salt that vary between
individuals. Some of these salts may act as a buffer when a
diagnostic assay demands operation at a pH different than that of
the urine sample. For example, urine sample with salts such as
bicarbonate, acetate or phosphate tend to resist the lowering of
pH. Creatinine, a common component of urine, may also act as a
buffer in resisting the lowering of pH. The result of this
buffering is that some urine samples will cause a strip pH to be
higher than optimal. A higher than optimal strip pH may result in
color generation of the protein indicator dyes, providing a false
indication of the presence of protein. It is desired to have a
buffing system that reduces such urine effects, while not
interacting with the chemistry occurring.
[0005] Accordingly, a need exists for a method and device for
detecting proteins over a wide range of clinical concentrations and
pH's.
SUMMARY OF THE INVENTION
[0006] According to one embodiment, an assay for the determination
of protein in an aqueous test fluid comprises combining the test
fluid with a buffer and a dye. The buffer is selected from
citrulline, malonic acid, cyanoacetic acid, citraconic acid, methyl
phosphonic acid, sarcosine, saccharin, or combinations thereof. The
buffer is added in sufficient quantity to maintain the pH of the
assay including the test fluid at a selected target pH range within
a range of from about 2.0 to about 3.0. The dye has a pKa which
enables it to operate as a protein indicator at the target pH
range. The dye has an affinity for protein such that it will
provide a detectable response in the presence of greater than about
15 mg/dL protein, to thereby render the assay suitable for the
detection of total protein in the test fluid.
[0007] According to another embodiment, a dry device for use in
determining protein levels in a fluid test sample comprises an
absorbent material having absorbed therein a buffer and a dye. The
buffer is selected from citrulline, malonic acid, cyanoacetic acid,
citraconic acid, methyl phosphonic acid, sarcosine, saccharin, or
combinations thereof. The buffer is added in sufficient quantity to
maintain the pH of the assay including the test fluid at a selected
target pH range within a range of from about 2.0 to about 3.0. The
dye has a pKa which enables it to operate as a protein indicator at
the target pH range. The dye has an affinity for protein such that
it will provide a detectable response in the presence of greater
than 15 mg/dL protein upon contact between the device and the fluid
test sample, to thereby render the device suitable for the
detection of total protein in the fluid test sample.
[0008] According to a further embodiment, an assay for the
determination of protein in an aqueous test fluid comprises
combining the test fluid with a buffer and a dye. The buffer is
selected from citrulline, malonic acid, or combinations thereof.
The buffer is added in sufficient quantity to maintain the pH of
the assay including the test fluid at a selected target pH range
within a range of from about 2.0 to about 3.0. The dye has a pKa
which enables it to operate as a protein indicator at the target pH
range and which has an affinity for protein such that it will
provide a detectable response in the presence of greater than about
15 mg/dL protein, to thereby render the assay suitable for the
detection of total protein in the test fluid.
[0009] According to yet a further embodiment, a dry device for use
in determining protein levels in a fluid test sample comprises an
absorbent material having absorbed therein a buffer and a dye. The
buffer is selected from citrulline, malonic acid or combinations
thereof. The buffer is added in sufficient quantity to maintain the
pH of the assay including the test fluid within a selected target
pH range within a range of from about 2.0 to about 3.0. The dye has
a pKa which enables it to operate as a protein indicator at the
target pH range and which has affinity for protein such that it
will provide a detectable response in the presence of greater than
15 mg/dL protein upon contact between the device and the fluid test
sample, to thereby render the device suitable for the detection of
total protein in the fluid test sample.
DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENT
[0010] The assay for protein of the present invention can be
carried out in either the wet or dry format. It is most
conveniently carried out in the form of an absorbent test strip
impregnated with (a) a buffer selected from citrulline, malonic
acid, cyanoacetic acid, citraconic acid, methyl phosphonic acid,
sarcosine, saccharin, or combinations thereof, and (b) a dye having
a pKa which enables it to operate as a protein indicator (e.g., a
color indicator) at a pH of from about 2.0 to about 3.0.
[0011] Buffers
[0012] The buffer to be used in the present invention is added in
sufficient quantity to maintain the pH of the assay including the
test fluid within a range of from about 2.0 to about 3.0. The
buffer may have a prior adjustment of pH that is commonly
accomplished by adding acid or base to a solution of the buffer
chemical to achieve a particular pH, or by adding a mixture of the
buffer chemical and its salt in selected proportions to give a
particular pH. The buffer is preferably added to maintain the pH of
the assay including the test fluid within about +/-0.2 pH units of
the selected target pH. The buffer is preferably added in
sufficient quantity to maintain the pH of the assay including the
test fluid within a range of from about 2.4 to about 3.0.
[0013] The preferred buffers are citrulline, malonic acid or
combinations thereof. Buffers may be selected from citrulline,
malonic acid, cyanoacetic acid, citraconic acid, methyl phosphonic
acid, sarcosine, saccharin, or combinations thereof. Various
concentrations of these buffers may be used, but the concentrations
of the buffers are generally from about 200 to about 500 mM.
[0014] Citrulline may be in the form of L-citrulline, D-citrulline
and D,L-citrulline and is represented by structure A. 1
[0015] Malonic acid that is useful in the present invention is
represented by Structure B, 2
[0016] Cyanoacetic acid that is useful in the present invention is
represented by Structure C, CO.sub.2H. t, Citraconic acid that is
useful in the present invention is represented by Structure D.
[0017] Structure D
[0018] Methyl phosphonic acid that is useful in the present
invention is represented by Structure E. environment than when free
in solution, and effectively develop a lower pKa for the ionization
essential to color change. If the dye, in the absence of a protein,
also responds as a pH sensitive color indicator within the selected
target pH range, then added proteins will generally not be detected
accurately.
[0019] The dyes to be used in the present invention generally
provide a detectable response in the presence of total protein from
about 2 to about 500 mg/dL protein. The dyes preferably provide a
detectable and particularly useful response to total protein from
about 15 to about 300 mg/dL protein. It is contemplated that the
amount of dye may be varied to give a detectable and useful
response outside of such a preferred range.
[0020] Examples of these dyes include substituted
phenolsulfonephthaleins, pyrogallol red or combinations thereof. It
is contemplated that other dyes that tend to have a detectable
indication in the dye absorbance or fluorescence spectrum (as
indicating transition) in the pH range of from about 2 to about 3
in the absence of protein and a different indication, under
otherwise the same conditions, but in the presence of protein may
be used in the present invention. The pH for giving a different
detectable response between the presence and absence of protein,
should be within the pH range maintainable by the buffer being used
in the present invention.
[0021] For example, it is contemplated that dyes such as
bromochlorophenol blue
(3',3"-dibromo-5',5"-dichlorophenolsulfonephthalein); bromophenol
blue (tetrabromophenol blue); basic fuchsin (basic red 9); basic
violet 14; martius yellow (acid yellow 24); phloxine B (acid red
92); methyl yellow (solvent yellow 2); congo red (direct red 28);
methyl orange (acid orange 52); and ethyl orange
(4-(4-diethylaminophenylazo)benzenesulfonic acid) may be useful in
the present invention. Such dyes may be used in combination with
dyes such as phenolsulfonephthaleins and pyrogallol red.
[0022] The phenolsulfonephthalein indicators particularly useful in
the present invention are represented by the following structures,
H and I: 3
[0023] Structure H represents the structure of a
phenolsulfonephthalein derivative in protic solvents such as water
or an alcohol, while Structure I represents the form that
predominates in the dry state or in aprotic solvents such as ethers
and acetonitrile. The phenolsulfonephthalein "protein error"
indicators are pH indicators that include an ionizable proton
having a pKa value such that at particular pH's the proton is not
ionized unless in the presence of protein.
[0024] The pKa value of a general phenolsulfonephthalein indicator
shown below, Structure J, is the pH at which one half of the number
of indicator molecules include the deprontated C ring phenolic
hydroxyl group. In the case of the phenolsulfonephthalein protein
error indicators illustrated above, two deprotonation events occur
to cause an observable color change. The first deprotonation
removes the proton from the aryl sulfonic acid on the A ring to
yield the ion illustrated below as Structure J. 4
[0025] The pKa of this proton is less than one, resulting in the
ionization of this moiety at all useful pH values. This ionizable
group is also responsible for the aqueous solubility of these
compounds. The second deprotonation involves the release of a
proton from the C ring phenolic hydroxyl to yield the dianion as
shown in Structures K and L below. 5
[0026] With this type of protein error indicator, the second
deprotonization causes the observable color change that is
indicative of the presence of protein in the sample being tested.
The phenolsulfonephthalein protein error indicator is typically
applied to an absorbent matrix material along with a buffer to
provide an environment of constant pH, so that one can rely on the
color change being the result of the presence of protein rather
than the result of a pH change upon contact with the test
fluid.
[0027] Only those phenolsulfonephthalein protein error indicators
which have a second pKa allowing the second deprotonation, in the
presence of protein, to take place at a pH within a range of from
about 2.0 to about 3.0 are useful in the present invention. Those
phenolsulfonephthalein protein error indicators that have a second
pKa allowing the second deprotonation, in the absence of protein,
to occur outside of the maintained pH may be useful in the
invention. Preferably, the second pKa, in the absence of protein,
takes place at a pH not within the range of from about 2.4 to about
3.0 and, most preferably, at a pH above 3.0. It is contemplated
that an ionization sensitive to protein presence that also gives a
detectable indication (e.g., a color response) may be a first,
second, etc. deprotonation with a different assortment of ionizable
substituents.
[0028] Many phenolsulfonephthalein protein error indicators have
their second pKa in the appropriate range and are, therefore,
suitable for use in the present invention. Useful
phenolsulfonephthaleins include those that are substituted with
electron withdrawing and/or electron donating groups such as amino,
amido, aromatic, alkyl, hydroxyl, thio, carboxylic, alkoxy, ketoxy,
acetyl, halogen, nitro, and cyano on the A, B or C rings of
Structures J and L. It is contemplated that one or more of the
rings may still contain a hydrogen. The particular substituent(s)
and their positions on the dye molecule are not critical so long as
the resulting dye exhibits a pKa in the appropriate range, and
retains desirable physical properties, such as solubility, protein
affinity and color. Unsubstituted phenolsulfonephthaleins are not
suitable because their pKa's are not in the appropriate range. It
has been discovered that the octa substituted sulfonephthalein
indicators, i.e., those phenolsulfonephthalein derivatives that are
substituted at the 3', 3", 5', 5", 3, 4, 5 and 6 positions, are
particularly suitable for use in the present invention. These
indicators are preferably substituted with halogen and nitro groups
and may be represented by Structure M: 6
[0029] where: X is nitro and Y and Z are chlorine, bromine or
iodine. Also useful are the nitro substituted polyhalogenated
phenolsulfonephthaleins disclosed in U.S. Pat. No. 5,279,790
wherein Y is nitro and X and Z are chlorine, bromine or iodine.
These indicators are said to have the ability to detect from about
2 to 500 mg/dL of protein in a fluid test sample. However, use of
the assay system of the present invention would be desirable in
situations in which proteinuria greater than 30 mg/dL is measured
and microalbuminuria at 2 mg/dL is not measured.
[0030] For example, in the case of diagnosing children for renal
disease, it is not desirable to measure microalbumin as this
response may give a false indication of disease due to its variable
nature. It is evident that the aromatic rings of the
phenolsulfonephthalein indicators useful in the present invention
can bear a variety of substituent groups. Such substituent groups
are limited only by the ability of one of ordinary skill in the art
to prepare stable compounds that have the appropriate protein error
indicator properties to render them suitable for use in the present
invention.
[0031] Some specific examples of the phenolsulfonephthalein dyes
are those substituted at the 5', 5" positions with nitro and at the
3', 3", 3, 4, 5 and 6 positions with chloride, bromide or iodide.
For example, phenolsulfonephthalein dyes that may be used include
5, 5"-Dinitro-3',3",3,4,5,6-hexabromophenolsulfonephthalein or
5'-Nitro-5"-Iodo-3',3",3,4,5,6,-hexabromophenolsulfonephthalein.
[0032] As discussed above, a dye that may be used is pyrogallol
red. Pyrogallol red typically includes the use of a molybdate or
tungstate salt that assists in providing rich color. U.S. Pat. No.
5,399,498 discloses that tungstate reacts with the indicator to
form a complex whose color shifts in the presence of protein in a
manner similar to that of molybdenum. U.S. Pat. No. 5,399,498 also
discloses the use of phytic acid or derivatives thereof to reduce
background interference in this sort of assay. While this assay is
very good at detecting low levels of protein in a fluid test sample
such as urine, its resolution drops off dramatically at higher
protein concentrations, particularly at protein concentrations
greater than about 150 mg/dL. This method is a total protein assay
since the response is not dependent on the type of protein present
in the test sample. Thus, human serum albumin (HSA) at 15 mg/dL
provides the same response as IgG at 15 mg/dL. Since total protein
assays are useful in the detection of certain disease states, and
the higher the total protein spillover into urine the more serious
the potential problem, a total protein assay that provides a
detectable response at high protein concentrations is desirable.
The molybdate/tungstate assay, however, is not effective for the
detection of high levels of protein, i.e. above about 150 mg/dL,
because of the strong affinity of the dye for protein.
[0033] Other Possible Components
[0034] The assay device or component may contain any number of
surfactants, detergents, background dyes, enhancer polymers or
chelating agents that are known in the art for use with protein
detection methods based on dye binding. These assay devices or
components typically include a protic solvent such as a
water/methanol mixture in the concentrations set out in Table 1.
Typically, the solution will contain a chelating agent such a
phytic acid and/or oxalic acid to inhibit or prevent interference
from other components in the urine test sample. The acids in the
paper dip process may be adjusted to a preferred pH of from about
2.4 to about 3 by adding sodium hydroxide or the like.
1TABLE 1 Optional Components Ingredient Function Conc. Used General
Range Water Solvent 95 mL -- Methanol Solvent 5 mL 0-40 mL Phytic
Acid Chelator 1 g (15 mM) 0-500 mM Oxalic Acid Chelator 0.11 g (12
mM) 0-40 mM
EXAMPLES
[0035] One example of forming a dry device, such as a diagnostic
strip, includes dipping a material such as paper into an aqueous
solution. The diagnostic strip may be made from various materials,
but is typically made from paper attached to a plastic carrier. One
example of a suitable paper is Ahlstrom 204. It is contemplated
that these strips may be made from other materials such as natural
or synthetic woven, or nonwoven materials.
[0036] According to one embodiment, a first aqueous solution is
formed by the addition of three submixes (Submixes 1-3). To form
Submix 1, 10 ml of methanol, 1.76 ml of 125 mM TRIS
((tris(hydroxymethyl)aminomethane)/metha- nol) and 44.0 mg of
pyrogallol red were added together. These components were mixed
from about 20 to about 30 minutes to form Submix 1. To form Submix
2, 4.10 g (3.20 mL) of 40% phytic acid, 8.00 mL of 5% aqueous PVA
(polyvinyl alcohol) of 31-50K, 11.2 ml of 1N NaOH and 0.654 mL of
100 mg/mL of sodium molybdate, and 45.6 mL of water were added
together. The NaOH was added to adjust the pH to approximately 2.3.
These components were mixed for about 60 minutes to form Submix 2.
To form Submix 3, 217.5 mg of disodium oxalate, 120.0 mL of 500 mM
L-citrulline with a pH of 2.5, and 100.0 mg of Cibacron Brilliant
Yellow were added together. The components were mixed for about 30
minutes to form Submix 3.
[0037] Submix 1 and Submix 2 were mixed together for about 10
minutes and then Submix 3 was added. The pH was 2.6 and the
combined Submixes 1-3 was stabilized for 4 hours and adjusted to
volume. Submixes 1-3 formed a First Dip solution of about 200 mL.
The paper was dipped into the First Dip solution and dried in a
three-stage oven at 50/50/70.degree. C. with a 1 inch air flow. The
paper being used was Ahlstrom 204 paper.
[0038] The paper was then dipped into a Second Dip solution of 160
mL. The Second Dip solution was made by mixing 144 mL of toluene,
16 mL of THF (stabilized), 0.48 g of KOK (a polypropyleneglycol
carbonate) such as disclosed in U.S. Pat. No. 5,424,215 and 36.57
mg of DNHB (5,5"
Dinitro-3',3",3,4,5,6-hexabromophenolsulfonephthalein). The paper
was dried in a three-stage oven at 50.degree./50.degree./50.degree.
C. with a 1 inch air flow to form the active pad of the diagnostic
strips. The diagnostic strips include the active pad that is
adhered to a polymeric strip.
[0039] While particular embodiments and applications of the present
invention have been illustrated and described, it is to be
understood that the invention is not limited to the precise
construction and compositions disclosed herein and that various
modifications, changes, and variations may be apparent from the
foregoing descriptions without departing from the spirit and scope
of the invention as defined in the appended claims.
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