CA 02470724 2004-06-16
DEVICES AND METHODS FOR ISOLATING AND DETECTING
SPECIFIC SUBSTANCES IN COMPLEX MATRICES
Related Applications
s This application claims priority to United States Provisional Application
60/342,425 filed on December 20, 2001, the entirety of which is expressly
incorporated herein by reference. Additionally, this application is a
continuation in
part of copending United States Patent Application Serial No. 09/183,157 filed
on
October 30, 1998 which is a continuation in part of United States Patent
Application
1 o Serial No. 08/723,636 filed on October 2, 1996, now United States Patent
No.
5,958,714 filed on Oct. 2, 1996. Copending United States Patent Application
Serial
No. 09/183,157 and issued United States Patent No. 5,958,714 are also
expressly
incorporated herein by reference.
15 Field Of The Invention
The present invention pertains generally to methods and apparatus for
analytical chemistry, and more particularly to test kits and methods for
qualitatively or
quantitatively determining one or more analytes present within a sample or
matrix.
2 o Background Of The Invention
It is routinely desirable to test for the presence of specific analytes in
substances which are intended for human consumption or application to the
human
body (e.g., foods, beverages, cosmetics, toiletries, topical solutions,
contact lens
solutions, pharmaceutical preparations, etc.) to confirm that such substances
are
2s fresh (i.e., not degraded), pure and free of contamination. Additionally,
it is often
desirable to test for the presence of specific analytes in samples of
biological fluids
(e.g., blood, plasma, serum, urine, saliva, bile, lymph, etc.) which have been
extracted from the human body.
However, the analytical techniques which have heretofore been utilized to
3 o quantitatively or qualitatively test for specific analytes in complex
matrices are often
problematic, due to the fact that such substances may contain many diverse
physical
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and/or chemical species, some or all of which may interfere with the intended
analysis. Thus, it is frequently necessary for the test substance to be
subjected to
extensive sample preparation steps, in order to isolate and/or concentrate the
particular analyte(s) of interest, prior to actually proceeding with
analytical
determination of the desired analyte(s). Moreover, in instances where the test
substance is a solid material (e.g., food) it is often necessary to chop or
grind the
solid material into particles, and to extract the desired analyte(s) from such
particles
by adding one or more liquid digestants, solvents or other fluids to form a
slurry or
suspension, and thereafter performing a "clean up" of the slurry or suspension
by
to filtration or centrifugation to separate the analyte containing liquid from
the
extraneous solid matter.
In instances where multiple analytes are to be determined, it is often
necessary to perform several separate, time consuming, analytical procedures
(e.g.,
gas chromatography (GC), high performance liquid chromatography (HPLC) or
other
analytical chemistry procedures) on aliquots or extracts of the test
substance, in
order to generate the desired multiple analyte data.
Thus, the traditional methods for determining the presence of, or detecting
specific analyte(s) in complex matrices (e.g., substances which contain matter
other
than the desired analyze(s)) can be quite time consuming, skill intensive and
2 o expensive.
It is frequently desirable to detect or quantify, in foods, one or more
particular
analyte(s) which are indicative of the freshness or quality of the food. In
routine
quality control testing of foods, it is common practice to test for the
presence of
various contaminates, additives, degradation products, and/or chemical markers
of
microbial infestation (e.g., bacterial endotoxins, mycotoxins, etc.). However,
the
current methods by which such quality control testing of food is accomplished
are
typically either: a) complex and skill-intensive analytical chemistry
procedures or b)
highly subjective and qualitative sensory evaluations (e.g., smell test, taste
test,
appearance, etc.).
3 o The quantities of certain food additives may be subject to governmental
regulation, especially in formulations wherein synthetic additives are being
utilized.
Thus, in such situations, it is typically desirable to perform chemical
analyses as
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means of determining the minimum amounts) of particular antioxidant additives
which must be added to a particular formulation to provide the desired effect
and/or
to identify non-regulated natural alternatives to governmental regulated
synthetic
additive. Thus, the detection and/or analysis of certain additives in foods
and other
s formulations is often carried out for various product/formulation
development or
research purposes, as well as for quality control testing of the freshness and
wholesomeness of the food or other product.
Also, bacterial or microbial contamination of foods and other substances is an
ongoing problem in a number of industries. In many instances, microbiological
to culture techniques are used to test for the presence of undesirable
microbial
contaminants in foods and other substances. These microbiological culture
techniques often take several days to complete and are subject to human error.
While PCR and other genetic techniques have been developed to quickly test for
the
presence of specific microbial DNA or RNA, the use of those techniques can be
15 problematic when the suspected microbial contamination is contained within
a food
or other complex matrix. Thus, there remains a need for the development of new
techniques for rapidly separating or isolating microbial DNA or RNA from a
complex
matrix such as a food and to thereafter detect the presence of such microbial
DNA or
RNA without the need for time consuming and laborious microbiological
culturing.
2 o In view of the foregoing problems and because the previously-known
analytical methods for determining specific analytes in relatively complex
matrices
(e.g., foods, biological fluids, etc.) may be too complex or too skill-
intensive for
untrained personnel, there exists a need in the art for the development of
simple test
kits capable of rapidly and reproducible determining the presence andlor
2 s concentrations of certain analytes or the presence of certain nucleic acid
sequences
in complex matrices, so that relatively untrained -personnel may pertorm such
determinations in a reliable, cost effective manner.
Some of the shortcomings of the prior art were overcome by the inventions
described in Applicant's copending United States Patent Application Serial No.
30 09/183,157 and previously issued United Stated Patent Nos. 5,958,714 and
6,489,123, the entireties of which are expressly incorporated herein by
reference.
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Summary of the Invention
The present invention provides methods and systems (e.g., test kits) for
qualitative and/or quantitative determination of one or more analytes present
within a
matrix that contains matter other than the analyte (e.g., solids, particulate
matter,
matter or substances that will interfere with the analysis, etc.). These
methods and
apparatus are useable to detect or quantify specific analytes present in
complex
matrices such as foods, cosmetics or biologicals, organ/tissue homogenates,
industrial waste, sewage, industrial fluids, microbiological or pharmaceutical
incubator slurries, etc. to determine the quality, degradation, age, abuse,
to contamination, nutritional value, purity and other characteristics of the
matrices.
In accordance with this invention, there is provided a method and system
(e.g., test kit) for determining the presence of a single analyte. This system
comprises; a) a sample receiving vessel, b) a membrane and c) a reagent-
containing
well. The test sample is initially prepared (e.g., chopped or ground if a
solid) and is
deposited in the sample-receiving vessel along with any desired diluent,
digestion
solution (e.g., enzymes), chelators, or chemical modifiers (e.g.,
antioxidants). The
prepared sample is then permitted to drain from the sample-receiving vessel,
through the membrane. The type of membrane utilized in each embodiment will be
selected based on the type and quantity of matter which is desired to be
excluded
2 o from the prepared sample matter prior to analysis. In many applications,
this initial
membrane will be formed of microporous film having pores which are sized to
present large particles of solid matter, proteins and other unwanted matter
from
passing therethrough, but which will allow a filtrate containing the desired
analyte to
drain into the reagent-containing well. When drained into reagent-containing
well, the
analyte contained within the filtrate will react with the reagent in a manner
which will
permit the presence or quantity of analyte to be determined. In many
instances, the
analyte-reagent reaction will be a color forming reaction such that a visual
determination may be made as to whether, or to what degree the desired analyte
is
present. In other instances, it may be desirable to utilize an analytical
instrument to
3 o determine the quantity of analyte present in the analyte present in the
analyte-
reagent solution. Examples of specific apparatus that may be used to support
the
membranes, facilitate flow of the sample/filtrate through the membranes and
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collection of the filtrates) and eluants for subsequent analysis are found in
copending United States Patent Application Serial No. 09/183,157 and
previously
issued United Stated Patent Nos. 5,958,714 and 6,489,123, the entireties of
which
are expressly incorporated herein by reference.
Further in accordance with this invention, a method or system of the above-
described character may be adapted for determination of two or more analytes
by
the addition of one or more additional membranes in series with the first
membrane.
Each of these additional membranes is operative to capture and hold at least
one
analyte, while allowing a filtrate containing one or more other analyte(s) to
pass
1o therethrough. Each of these additional membranes may subsequently be
exposed to
a wash or flush solution such that one or more eluants containing each of the
additional analytes may be obtained. Each such eluant may subsequently be
combined with a reagent to provide an eluant-reagent admixture from which at
least
one analyte may be determined. In this manner, the present invention is
adaptable
for the qualitative or quantitative determination of two or more analytes from
a single
sample.
Further in accordance with this invention, in situations where one or more
analytes is/are present in a matrix at low concentrations (e.g.,
concentrations that
are below the detection limit of the intended analytical test) the analyte may
be
2 o captured on a membrane and may be subsequently eluted from that membrane
with
a volume of eluent that is substantially smaller than the volume of the
original
sample, thereby providing an analyte/eluant admixture wherein the
concentration of
the analyte is sufficiently high to permit its detection by the intended
analytical
method. The starting concentration of the analyte in the original sample may
then be
determined by calculation based on the known volume of the original sample and
the
known volume of the eluant that was used to elute the analyte from the
membrane.
Further in accordance with this invention, there are provided methods and
systems of the foregoing character wherein a membrane is used to remove a
positive or negative interferant from the sample to permit an analyte to be
analyzed
3 0 or detected by chemical or biochemical methods without interference. One
particular
embodiment of this invention wherein an analyte is removed comprises a method
and system wherein free fatty acids (FFA) are present in a sample (e.g., a
food or
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oil) along with one or more inorganic acids. The analytical method intended to
be
used to detect or to quantitate the presence of FFA will also detect the
presence of
inorganic acids. Therefore it is desired to remove the inorganic acids) from
the
sample prior to analysis for the FFA. To accomplish this, the sample is passed
through at least one negatively charged membrane that captures inorganic acids
but
allows a filtrate containg any FFA's present in the sample to pass
therethrough. The
FFA containing filtrate is then subjected to the analytical test for FFA's and
an
accurate quantitative or qualitative determination of FFA's is then obtained.
In some
situations it is additionally desired to qualitatively or quantitatively
analyze the
to inorganic acid that was present in the sample. In such situations, an
eluant that
releases the inorganic acid from the negatively charged membrane is used to
elute
the inorganic acid from the membrane on which it was captured, thereby
providing
an inorganic acid/eluant admixture from which the inorganic acid may be
quantitatively or qualitatively analyzed. In some situations it may be
additionally
desirable to desperate specific types of inorganic acids present in the sample
and to
analyze for one or both of those types of inorganic acids. Accordingly, in
such
instances, the sample may be passed through a plurality of membranes, each of
which has a binding affinity for a different type of inorganic acid, before
the filtrate is
analyzed for FFA. In this regard, a first membrane may be impregnated or
coated
2 o with a substance which carries a sufficient negative charge to bind weak
inorganic
acids (e.g., acetic acid) and a second membrane may be impregnated or coated
with
a substance which carries a sufficient negative charge to bind stronger
inorganic
acids (e.g., citric acid). The weak and strong inorganic acids that become
bound to
these membranes may then be separately eluted and analyzed, if desired. In
other
2 5 instances, it may be desirable to perform an enzymatic analysis for a
particular
analyte contained in a sample but the presence of metals in the sample may
interfere with such enzymatic analysis. In such instances, the sample may be
passed through an anionic membrane which will bind and hold metals present in
the
sample and the desired enzymatic analysis may then be performed on the metal
free
3 o filtrate without interference from the previously present metals.
Still further in accordance with this invention, there are provided methods
and
systems wherein the sample is passed through a membrane (e.g., a membrane that
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is impregnated or coated with specific antibodies) which binds certain amino
acid
sequences. The particular amino acid sequence may be selected on the basis of
its
known presence in the nucleic acid (e.g., DNA or RNA) of a particular organism
or
microbe (e.g., bacteria, virus, parasite, spore, prion, etc.), a genetically
modified
s substance or a protein, that may be present in the sample. The bound nucleic
acid(s), genetically modified substances) or proteins) are then eluted or
released
from the membrane and subjected to an analytical or detection technique, such
as
amplification and PCR, whereby a quantitative or qualitative determination of
that
nucleic acid or protein is made. This aspect of the invention is useable to
determine
Zo the presence or concentration of certain pathogenic or deleterious
microbes, toxic or
deleterious proteins, or the presence of a prohibited or regulated substance
(e.g.,
genetically modified plant substances or grain) in a food, beverage, water,
medicine,
cosmetic or other sample.
Still further in accordance with this invention, there are provided methods
and
15 systems wherein a sample is passed through a pre-weighed membrane which has
a
selective affinity to bind a certain substance. The membrane with the
substance
bound thereto is then reweighed to determine the weight of the substance that
was
present in the sample. In this regard, a food or beverage sample may be passed
through a membrane that has a specific binding affinity for proteins.
Thereafter the
2 o membrane (with the protein bound thereto) may be weighed and the weight of
the
protein removed from the sample may be calculated. On this basis, one may also
calculate the % protein present in the sample. Alternatively, the protein may
be
eluded from the membrane and analyzed as described herein.
Brief Description of the Drawings
2 5 Figure 1 is a schematic diagram of a single membrane device useable with
some of the methods and systems of the present invention.
Figure 2 is a schematic diagram of a plural membrane useable with some of
the methods and systems of the present invention.
Figure 3 is a table listing specific filtration and capture membranes that may
3 o be used in the present invention.
Figure 4 is a table listing specific detection reagents that may be used for
detection or analysis of analytes in the present invention.
CA 02470724 2004-06-16
Figure 5 is a table listing specific test methods and systems and specifying
the analytes, typical matricies in which the analyte is contained, specific
membranes
(cross-referenced to Figure 3) and the specific detection reagents (cross-
referenced
to Figure 4) useable in each test method and system.
Detailed Description
The following detailed description and the figures to which it refers are not
intended to describe all possible embodiments and examples of the invention.
Rather, this detailed description and the accompanying figures are directed to
certain
illustrative embodiments and examples of the invention only and does not limit
the
1 o scope of the invention in any way.
Methods And Systems of the Present Invention:
The present invention includes a number of specific methods and
systems (e.g., combinations of membranes, eluants and reagents; test kits)
that may
be used to obtain quantitative or qualitative determinations of specific
analytes in
15 foods, oils and other matrices. The methods and systems may be used in
conjunction with the devices described in copending United States Patent
Application Serial No. 09/183,157 and previously issued United Stated Patent
Nos.
5,958,714 and 6,489,123, the entireties of which are expressly incorporated
herein
by reference. Certain embodiments of these devices are commercially available
as
2 o the SaftestT"" Membrane Unit and the SaftestT"" Filtration Unit from
Saftest, Inc., 3550
North Central, Suite 1400, Phoenix, AZ 85012. Figures 1 and 2 show, in
schematic
fashion, examples of devices used in conjunction with the methods and systems
of
this invention.
Specifically, Figure 1 shows a single membrane device 10. This single
2 s membrane device 10 comprises a sample well 12, a membrane support 15, and
a
filtrate collection well 16. In embodiments where the sample 18 comprises
matrix
that includes a liquid phase wherein the analyte as well as extraneous matter
(e.g.,
solid particles or large molecular weight compounds) a filtration membrane 13
having
pores that are small enough to prevent passage therethrough of the extraneous
3 o matter but large enough to permit passage therethrough of the analyte-
containg
liquid phase is positioned on the membrane support. The sample 18 then passes
from the sample well 12 and through the filtration membrane 13, whereby the
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CA 02470724 2004-06-16
extraneous matter is retained above the membrane and a filtrate 16 containing
the
analyte passes through the filtration membrane 13 and into the filtrate
collection well
16. A desired analytical or detection technique may then be used to
quantitatively
or qualitatively determine the analyte in the filtrate 20. In some instances,
such
analysis will require one or more reagents to be mixed with the analyte-
containing
filtrate 20. In other instances, the neat filtrate 20 may be used for the
analysis (e.g.,
examined microscopically, placed in an analytical instrument such as a
spectrophotometer or chromatograph or applied to an indicator (e.g., pH paper,
paper or dip sticks which indicate the presence of the analyte, etc.) In other
1 o embodiments, the sample18 may be substantially free of extraneous matter
that
must be removed by a filtration membrane 13 (e.g., a clean oil or liquid
solution) but,
instead, the sample 18 may contain two analytes that must be separated or some
interferant that will interfere with analysis for the analyte and must
therefore be
separated from the analyte prior to analysis. In these embodiments, a capture
membrane 14 will be mounted on the membrane support rather than a filtration
membrane 13. This capture membrane 14 may be selected so as to capture (e.g.,
chemically bond to or otherwise hold) a first analyte while allowing a second
analyte
to pass therethrough in the filtrate 20. The first analyte may subsequently be
eluted
(e.g., released) from the capture membrane and determined separately and the
first
2 o analyte contained in the filtrate 20 may also be determined. The capture
membrane
may also be used to capture an interferant while allowing a filtrate containg
the
analyte to pass therethrough or vice versa.
Figure 2 shows, in schematic fashion, a two membrane device . Here, the top
membrane is either a filtration membrane 13 (for samples 18 which contain
extraneous matter that must be filtered out) or a capture membrane 14 (for
samples
that contain multiple analytes or interferants). The bottom membrane is a
capture
membrane 14. The sample 18 passes through the top membrane which removes
extraneous matter or captures a first analyte or interferant. The filtrate
that has
passed through the top membrane then passes through the bottom membrane which
3 o captures an analyte or interferant and the filtrate 20 that has passed
through both
membranes then collects in the filtrate well 20. An analyte contained in the
final
filtrate 20 may be determined as described above. If one or both of the
membranes
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have been used to capture another analyte(s), such other analyte(s) may be
eluted
from the membranes) and determined separately. In the example of Figure 2, it
shows that the bottom capture membrane 14 is transferred to a second membrane
support 15a. An eluant 22 is then passed through the capture membrane 14 so as
to elute (e.g., release) the analyte from that membrane 14. An eluant/analyte
admixture 24 is then collected in a collection well 26. The second analyte may
then
be quantitatively or qualitatively determined from the eluant/analyte
admixture. As
summarized above, in some embodiments, it may not be necessary to elute the
second analyte from the membrane. Rather, the membrane may contain an
to indicator that changes to indicate the presence of the analyte thereon or
the
membrane may be weighed to determine the weight of the analyte contained
thereon.
As explained in incorporated United States Patent Serial No. 6,489,123, and
copending parent application Serial No. 09/183,157 more than two, and
virtually any
i5 number, of membranes 13, 14 may be used to capture and optionally analyze
virtually any number of analytes or inerferants.
Examples of the filtration membranes 13, capture membranes 14 and
reagents useable for specific embodiments of the present invention are shown
in the
tables of Figures 3, 4 and 5. Specific embodiments of the present invention
include
2 o the following:
1. A method and system for citric acid and free fatty acid determinations in
any
sample, for example, an oil. The sample (e.g., oil) is passed through a
positively
charged anionic membrane for capture of the citric acid from the oil and
detection of
2 s free fatty acids in the filtrate. The citric acid that has become bound to
the positively
charged anionic membrane is then eluted or released from the membrane using a
high salt solution (e.g., 0.5 M NaCI in water) as the eluant. The
eluant/citric acid
admixture is then combined with sulfanilic acid hydrochloride with a nitrite
activator
(e.g., 0.2% sulfanilic acid and 5% sodium nitrite). This results in a color
reaction
3 o indicative of the presence of citric acid. In addition to oils, this
citric acid/free fatty
acid system can be used for determinations in various other matrices including
food.
In foods which contain encapsulated lipids, the food may be soluablized such
that
to
CA 02470724 2004-06-16
the lipids are dissolved in a liquid phase. A first membrane may be used to
remove
solid extraneous matter. The liquid, lipid-containing filtrate is then passed
through
the capture membrane such that the citric acid becomes bound to the capture
membrane. The free fatty acids are then measured in the filtrate that passes
through
the capture membrane. The citric acid is then released from the capture
membrane
by elution with a salt solution as described above. The eluant/citric acid
admixture
may then be contained in a second vessel and the presence and/or amount of
citric
acid may be analyzed as described above.
2. A method and system for determining acetic acid and free fatty acid can be
used for determinations in other food matrices and encapsulated lipids in
foods
where the food is solubilized. In the same manner as the citric acid assay
described
above, a first filtration membrane is used to remove particles and other solid
matter.
The acetic acid containg bound to the capture membrane, the free fatty acids
z5 measured in the effluent and then the acetic acid released from the capture
membrane with high salt solution into a second vessel and quantitated.
4. Test kit for alkenal acid determinations in oil. A first filtration
membrane and
oil in food using a particulate removing filtration membrane and then a methyl
indole
2 0 or methylphenyl indole detection system with a very strong acid such as
methane
sulfonic acid.
5. Test kit for prediction of oxidative degradation of seafood using a
particulate
removing membrane and then malonaldehyde as a detector to quantitate indolic
2 5 compounds formed in the degradation of shrimp..
6. Any of the above kits s to be used in conjunction with a second test for
malonaldehyde utilizing a methyl indole reagent with weak acid such as small
amount of HCI.
7. Any of the above kits to be used with a second test for lipid peroxides in
the
eluant using an iron catalyzed electron transfer to xylenol orange.
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8. Any of the above kits to be used with a second test for Free Fatty acids
using
an alcoholic indicator such as isopropanol/xylenol orange.
s 9. A test kit for protein determination on filtered and unfiltered oils in
conjunction
with the citric acid determination by using one membrane to bind citric acid
and one
to bind protein and eluting each membrane separately and detecting the
analyte.
10. A test kit for protein determination on refined oil concentrating the
protein on a
1o protein binding membrane by passing 1 to 200m1 of oil through the membrane
and
eluting the protein off into another tube using a salt solution in 1 ml.
11. A test kit for protein determination in meals by digesting the meal with
phosphoric or another strong acid and using a particulate removing membrane to
15 remove debris and then testing the filtrate for protein.
12. A test kit for protein determination on tallows or greases by using a
membrane
to bind protein and eluting the membrane and detecting the analyte.
2 0 13. A test method and kit for determination of polymerized and non-
polymerized
oils in cooking fats and oils in conjunction with the alkenal determinations
on filtrate
to determine frying oil quality and oil. Quality in fried foods using a
molecular weight
cutoff membrane to capture the polymerized lipids and then release them to
measure triglyceride content.
2s
14. A test method and kit to determine oxidation of beverages and
determination
efficacy of certain additives and/or stabilizers on oxidation using the
alkenal test .The
beverage, carbonated or not, is separated through protein binding membrane and
the filtrate tested with methylindolis solution with sulfonic acid.
15. A test for rapid determination of the quality of cooking oils and fats by
testing
for lipid peroxides using a peroxidase and iron catalyzed reagent and
complexed to
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xylenol orange and alkenals using the methylindole reagent with a very strong
acid
added This test can be used on beer or beverages and predict quality and shelf
life
of beverages.
s 16. A test method and kit to detect specific microbes or viruses in foods or
tissues
by emulsifying the food and releasing the nucleic acids using surfactants or
osmotic
changes to lyse the membranes and cells and using a particulate binding
membrane
followed by a nucleic acid binding membrane. The DNA is released and then
amplification of a sequence specific to the target organism to detect its
presence
1 o performed.
17. A test method and kit to detect aflatoxins in foods or tissues by
emulsifying
the food and releasing the aflatoxins using surfactants or osmotic changes to
lyse
the membranes and cells and after filtering out particulates using a second
15 membrane coated with an antibody specific to multiple or particular
aflatoxins. The
aflatoxins are released and then detected using peroxidase conjugated
antibodies
18. A test method and kit to detect specific live microbes or viruses in foods
or
tissues by emulsifying the food and releasing the nucleic acids using
surfactants or
2 0 osmotic changes to lyse the membranes and cells and using a particulate
binding
membrane followed by a ribonucleic acid binding membrane. The RNA is released
and then amplification of a sequence specific to the target organism to detect
its
presence performed.
Detailed Examples Of Specific Embodiments Of The Present Invention
3 o The following examples demonstrate methods of detecting various analytes
contained in samples, in accordance with the invention disclosed hereinabove.
The
analytes may be removed from a sample using a device or system incorporating
one
or more membranes for filtering the sample, such as devices and systems
disclosed
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in commonly owned PCT International Patent Publication No. WO 99/20396 and
U.S. Patent No. 6,489,132, and the publicly available SafTestT"" Filtration
Unit
available from Saftest, Inc. (Phoenix, AZ). PCT International Patent
Publication No.
WO 99/20396 and U.S. Patent No. 6,489,132 are expressly incorporated herein by
s reference.
Example 1
Separation and Determination of Free Fatty Acid (FFA) and Citric Acid
in an Oil Sample
This example demonstrates free fatty acids contained in an oil
sample. The oil sample also contains citric acid. It is desirable to
separate the citric acid from the sample prior to assay of the FFA
content as the presence of inorganic acids such as citric
A 1 mL sample of soybean oil is applied to a membrane of a
filtering device. The membrane is a strongly basic anionic membrane,
such as the Q membrane adsorber membrane with quaternary
ammonium groups (Q-MA membrane) publicly available from Sartorius
(Sartorius North America, Inc., Edgewood, NY). As the sample is
2 o applied to the membrane, the citric acid is retained by the membrane,
and the remaining oil containing free fatty acids is collected in a
container.
The membrane containing the citric acid is removed from the
container and is washed with 1 mL of 0.5 M NaCI in water. The eluant
2 s is collected in a second container. One mL of the eluant containing
citric acid is mixed with 0.3 mL of a reagent containing 0.2% sulfanilic
acid and 5% sodium nitrite. The reaction occurs for about 30 minutes
an elevated temperature (approximately 42-45 °C). The presence of
citric acid in the sample results in a yellow color which can be
3 o measured by examining the reaction mixture with a spectrometer at
420 nm, and comparing the calculation to one or more standards. A
test kit suitable for performing this citric acid assay is commercially
available under the name CitriSafeTM from Saftest, Inc. (Phoenix, AZ).
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The CitriSafeT"" test kit is generally described in Appendix A to this
patent application.
The amount of free fatty acids originally present in the soybean
oil is determined by measuring the acidity of the oil after the removal of
the citric acid using the methodology described in incorporated parent
application Serial No. 09/183,157 and commercially available as a test
kit under the name FASafeT"" from Saftest, Inc. (Phoenix, AZ).
The CitriSafeT"" and FASafeT"" test kits are useable in
conjunction with devices described in copending United States Patent
1o Application Serial No. 09/183,157 and previously issued United Stated
Patent Nos. 5,958,714 and 6,489,123, the entireties of which are
expressly incorporated herein by reference. Certain embodiments of
these devices are commercially available as the SaftestTM Membrane
Unit and the SaftestT"" Filtration Unit or SaftestT"" Work Station, from
Saftest, Inc.
Example 2
Removal of Inten'eriny Inorganic Acids and Determination of
Free Fafty Acid (FFA) and Acetic Acid in a Food Sample
2 o This example demonstrates the separation of inorganic acids
from free fatty acids in a food sample and the subsequent
determination of acetic acid and free fatty acid content of that food
sample.
A 5 gram sample of mackerel is solubilized to create a slurry.
The slurry is heated to approximately 40-45 °C and filtered to
remove
particulates from the slurry. Two (2) mL of the filtered slurry is applied
to a membrane structure of a filtering device. The membrane structure
includes two stacked membranes one disposed on top of the other.
The upper membrane is a weakly basic membrane, such as the D
3 o membrane adsorber with diethylamine groups (the MA-D membrane),
and the lower membrane is a strongly basic membrane, such as the
membrane used in Example 1. These membranes are publicly
available from Sartorius (Sartorius North America, Inc., Edgewood,
NY). As the filtered slurry is applied to the membrane structure, acetic
is
CA 02470724 2004-06-16
acid, and other weak inorganic acids, are retained by the upper
membrane, and citric acid, and other strong inorganic acids are
retained in the lower membrane. The remaining slurry containing free
fatty acids is collected in a container.
s The membrane containing the acetic acid is removed from the
container and is washed with 2 mL of 1 M NaCI in water. The eluant is
collected in a second container. 100 pl of the eluant containing acetic
acid is mixed with 1.0 mL of a reagent containing 0.1 % xylenol orange
in neutralized isopropanol. The reaction occurs for about 10 minutes at
1 o an elevated temperature (approximately 42-45 °C). The presence of
acetic acid in the sample is determined by examining the reaction
mixture with a spectrometer at 570 nm. The amount of acetic acid
present in the sample is determined by comparing the results to one or
more standards.
15 The citric acid is removed from the lower membrane using the
procedure disclosed in Example 1.
The amount of free fatty acids originally present in the fish slurry
is determined by measuring the acidity of the oil after the removal of
the inorganic acids using the FASafeT"" publicly available from Saftest,
2 o Inc. (Phoenix, AZ). The FASafeT"" test kit is useable in conjunction with
devices described in copending United States Patent Application Serial
No. 09/183,157 and previously issued United Stated Patent Nos.
5,958,714 and 6,489,123, the entireties of which are expressly
incorporated herein by reference. Certain embodiments of these
25 devices are commercially available as the SaftestT"" Membrane Unit
and the SaftestT"" Filtration Unit or SaftestT"" Work Station, from Saftest,
Inc.
16
CA 02470724 2004-06-16
Example 3
Determination of Total Fat Content and/or Percent Fat
This example demonstrates the determination of fat content or
s the percent of fat in foods.
Eight ounces of salad dressing is heated and homogenized with
stabilized 100% isopropanol to release lipids in the salad dressing that
are bound to proteins or held in membranes of items in the salad
dressing. The homogenate is prefiltered to remove particulates using a
1 o cellulose acetate membrane having a pore size of 0.45 microns. The
filtered homogenate is passed through a membrane that binds
proteins, such as the polyethersulfone (PES) membrane sold by
Sartorius, Inc, and then the filtered homogenate is passed through a
membrane that binds surfactants, such as the MA-Q or MA-S
15 membranes from Sartorius. The MA-S membrane has sulfonyl groups
on the membrane surface for binding surfactants.
A portion of the filtrate (20 uL) that is free of proteins and
surfactants is mixed with 1.0 mL of lipase (Sigma, St. Louis, MO) in
phosphate buffer to enzymatically cleave the fatty acids from glycerol.
2 o The amount of glycerol present in the filtrate is measured enzymatically
using a series of enzyme reactions using glycerol kinase and ATP to
produce glycerol 1-phosphate and glycerol-1 phosphatase to produce
dihydroxyacetone, which is detected with a peroxidase catalyzed
reaction with aminoantipyrine to produce a measurable quinoneimine
25 dye. This reaction is complete in 10 minutes at 42 °C. By measuring
the amount of glycerol present in the filtrate, the total fat content
contained in the salad dressing is determined without regard to the
specific proportions of the various proportions of free fatty acids.
A test kit for this percent fat assay is commercially available as
3 o Percent Fat Kit MSA from Saftest, Inc. (Phoenix, AZ) and is described
in Appendix B to this patent application. The Percent Fat Kit MSA is
useable in conjunction with devices described in copending United
States Patent Application Serial No. 09/183,157 and previously issued
m
CA 02470724 2004-06-16
United Stated Patent Nos. 5,958,714 and 6,489,123, the entireties of
which are expressly incorporated herein by reference. Certain
embodiments of these devices are commercially available as the
SaftestTM Membrane Unit and the SaftestT"~ Filtration Unit or SaftestT""
s Work Station, from Saftest, Inc.
Example 4
Determination of Protein Content in Refined Oil
Zo In this example the present invention is used to determine total
protein content in a refined oil such as soy bean oil.
A 5 mL sample of refined and genetically modified soy bean oil
is heated to approximately 40 °C and is mixed with 5 mL of 100%
isopropanol. The warm mixture is applied to a membrane that binds
15 proteins, such as the membrane used in Example 1. The protein in the
oil/alcohol mixture binds to the membrane, and the fatty acids
contained in the mixture pass into a container.
The protein-containing membrane is moved to another container
and is washed to release the protein into the container with 1 mL of
2 o buffered, low salt solution (0.05 M NaCI in phosphate buffer at a pH
between 7 and 9). One (1 ) mL of the concentrated filtrate is mixed with
0.3 mL of an indicator solution containing 0.1 % brilliant blue (Sigma, St.
Louis, MO) in 30% methanol, and 0.3% phosphoric acid for two
minutes at room temperature (18-25 °C). The presence of protein is
2 5 qualitatively determined by the presence of a blue color in the mixture.
The amount of protein is quantified by comparing the blue color of the
mixture to one or more standards, and/or by using a spectrometer at
570nm.
A test kit for this protein content assay is commercially available
3 o as ProteSafeT"" from Saftest, Inc. (Phoenix, AZ) and is described in
Appendix C to this patent application. The ProteSafeT"~ test kit is
useable in conjunction with devices described in copending United
is
CA 02470724 2004-06-16
States Patent Application Serial No. 09/183,157 and previously issued
United Stated Patent Nos. 5,958,714 and 6,489,123, the entireties of
which are expressly incorporated herein by reference. Certain
embodiments of these devices are commercially available as the
s SaftestT"" Membrane Unit and the SaftestT"" Filtration Unit or SaftestT""
Work Station, from Saftest, Inc.
Example 5
Determining the Presence Of A Particular Microbe in a Food Sample
1 o This example demonstrates methods to identify the presence of
one or more microbes, including pathogenic and non-pathogenic
bacteria and viruses, in food products. The microbes are detected by
binding nucleic acids to one or more membranes, and amplifying the
nucleic acids using nucleic acid primers having a desired nucleotide
is sequence for the microbes.
Ten grams of ground beef is prepared for determination of the
presence of ecoli H157. The ground beef is homogenized with a
buffered solution, such as phosphate buffer, containing 1-2% sodium
dodecyl sulfate (SDS) in a ratio of approximately 1 to 4, of beef to
2 o diluent, to disrupt the cellular component and to release nucleic acids
contained within the beef. The slurry of homogenized ground beef is
applied to a first membrane, such as a polytetraflouroethylene
membrane (available from Sartorius), to remove particulates from the
ground beef. The filtered slurry is then applied to a second membrane
2 s that is configured to bind DNA or RNA. Anionic membranes, such as
MA-Q membranes from Sartorius, or membranes having one or more
types of nucleic acid binding antibodies, such as MA-A membranes
(Sartorius), which has crosslinked antibodies attached to it by
glutaraldehyde crosslinking, or the MA-I Iminodiacetic acid membranes
30 (Sartorius), which are reacted with the protein amino groups of the
antibodies. The slurry is passed through two additional nucleic acid
19
CA 02470724 2004-06-16
binding membranes to increase the amount of nucleic acid removed
from the homogenate. The filtrate is then discarded.
The membranes are washed with 1 M NaCI in water to release
the nucleic acids from the membranes into a container. The RNA and
DNA are then amplified using polymerase chain reaction (PCR) and
one or more nucleic acid primers that have sequences for ecoli H157.
PCR methods are conventionally known to persons of ordinary skill in
the art, see, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 3~d Edition, 2001. The PCR products are labeled
to by incorporating a fluorescent marker during the amplification steps,
and the presence of ecoli H157 is determined by measuring the
fluorescence contained in the PCR products.
Although exemplary embodiments of the invention have been shown and
described above, many changes, modifications, substitutions, variations and/or
additions may be made by those having ordinary skill in the art without
necessarily
departing from the spirit and scope of this invention. For example, where this
patent
application has described the performance of steps of a method or procedure in
a
specific order, it may be possible (or even expedient in certain
circumstances) to
2 o change the order in which some steps are performed, and it is intended
that the
particular steps of the method or procedure claims set forth herebelow not be
construed as being order-specific unless such order specificity is expressly
stated in
the claim. Another example is that, although specific membranes and reagents
are
called out above, various other membranes or reagents or equivalent materials
may
2 s be used to bring about the same or substantially the same effects as
described
herein and those other membranes and reagents may also be useable to practice
the methods of the present invention. Accordingly, it is intended that all
such
changes, modifications, substitutions, variations and/or additions be included
within
the scope of the following claims.
CA 02470724 2004-06-16
APPENDIX A
ft~~iS~frE~ ~'l STD Kit Assay Instructions
Standard Kit
Introduction
The Safety Associates, Inc. CifriSafe'"" STD assay measures the citric acid
concentration in samples as parts per
million (ppm).
Dispenser and Reagent Preparation
Allow reagents and dispensers to stand at room temperature (98-25°C)
for 15-20 minutes before beginning
the assay.
1.' Mix the contents of the reagent bottles by gently swirling prior to
attaching dispensers.
2. Attach the dispenser labeled in yellow as "CitriSafe Elution Buffed' onto
the 500 mi reagent bottle labeled
"CitriSafe Elution Buffer" and set the volume to 750 pl.
3. Attach the dispenser labeled in yellow as "CitriSafe A" onto the 500 ml
reagent bottle labeled "CitriSafe
Reagent A" and set the volume to 1.5 ml.
4. Attach the dispenser labeled in yellow as "CitriSafe B" onto the 250 mi
reagent bottle labeled "CitriSafe
Reagent B ° The volume is fixed at 100 pl, so no adjustment is
necessary.
5. Dispense each reagent into a waste container until there are no air bubbles
in the dispensers
(approximately 2-5 aliquots, slowly).
Sample Preparation
Refer to the Sample Preparation Section of this manual for detailed
instructions.
Pre-Assay Preparation
1. Label a new set of test tubes: RB for the reagent blank, C1 through C3 for
the calibrators, and 1,2,3 etc.
for
the samples. If running duplicates, label two test tubes for each sample.
Also, label the yellow Q-
Membrane Units (QMU) with the sample numbers.
2. Using the adjustable pipette, transfer 750 ui of the reagent blank and 750
pl of each calibrator into the
designated test tubes. Use a new tip for each standard and reagent blank.
Always wipe the pipette tip
prior to dispensing the reagent blank and standards.
3. Cap the test tubes to minimize evaporation and set aside in a test tube
rack until needed.
21
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
4. Set up the SatTestT"' Filtration Unit. Place the first six labeled test
tubes into the base. Attach a
membrane holder to the base, and then attach six-labeled Q-Membrane Units
(G1MU) to the membrane
holder. The QMU labels should correspond to the labels on the test tubes.
Uncap the QMU's.
Assay Performance
1. Using the positive displacement pipette, add 1 ml of oil to each QMU. Use a
new tip for each sarhple.
Always wipe the pipette tip prior to dispensing the samples. Recap the QMUs.
2. If testing hydrogenated samples, warm them until Ilquid and dilute 1:2.
3. Once the sample is diluted analyze as usual.
4. Tum on the SafTestT" Filtration Unit and filter the samples. A vacuum
should be evident on the vacuum
gauge at 8-10 psi. If there is no change in the gauge, recheck all seals.
5. Remove the MSU keeping the QMUs in place. Remove the test tubes containing
the filtrates. Label new
set of test tubes and place them in the Saf1'estT"' Filtration Unit. Reattach
the MSU and QMUs. Uncap
the QMUs:
6. Using the bottle-top dispenser, dispense 750 Irl of Elution Buffer to each
QMU. Tum on the SatTestT"
Filtration Unit and filter the samples at 1-2 psi.
7. After littering is complete and the vacuum pump is still on, carefully
unscrew the QMU to release the
vacuum and get the last drop of buffer into the tube.
8. Remove the MSU keeping the QMUs in place. Remove the test tubes and place
them In a test tube rack.
9. Dispense one aliquot (1.5 ml) Reagent A into the reagent blank tube, each
of the three standard tubes,
and each sample tube.
10. Dispense one aliquot (100p1) Reagent B into the reagent blank tube, each
of the three standard tubes,
and each sample tube.
1 i . Cap all of the tubes and vortex each for ten seconds. Place all tubes
(reagent blank, standards, and
samples) into a 42-45°C heat block. Set the timer for 30 minutes, and
allow tubes to heat up for 30
minutes. Vortex the samples for 5 seconds at the end of the incubation cycle.
12. Approximately 10 minutes before the end of the heating period, turn on the
SatTestT"" Analyzer and allow
it to warm up. Place the 4201680 filter in the fitter compartment. Select
"CITISTD" program.
13. After 30 the minute heating period, blank the SatTestT"' Analyzer by
inserting a water blank followed by
the reagent blank. If the reagent blank has an optical density (OD) reading
>0.0500 on the SaflestT""
Analyzer, it is unacceptable and must be repeated.
14. Insert Standards 1 through 3 as prompted by the SafTestT" Analyzer. Insert
the sample test tubes in the .
designated order. Wipe each test tube with a lint-free tissue prior to
insertion In the SatTestT"" Analyzer.
22
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Reporting Results
1. The SafTest""Analyzer will report results as parts per million (ppm) citric
acid in the sample.
2. If preliminary dilutions were made to the oil sample prior to testing, the
dilution factor must be taken into
account. Multiply the instrument results by the dilution factor to obtain the
value of the sample.
3. If the sample value is greater than the value of the highest calibrator,
the instrument will flag the results
as "HI " The sample must be diluted to a higher dilution and retested.
4. If the sample value is less than the value of the lowest calibrator, the
instrument will flag the results as
"LO" and should be reported as "< (value of the lowest calibrator) °
5. Check the instrument printout for flags or error messages before reporting
results. A large coefficient of
variation (ACV) is expected with samples that are measured at the low end of
the calibration curve.
6. For failed curve fit (r= < 0.990), repeat the assay.
7. Ranges for the controls are found on the package insert provided in the
Control Kit. Assay values for the
controls should approximate these ranges. However, it is advised that each lab
establish control ranges
using their own equipment and personnel.
23
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Kit for Oils
Purpose The c2nSa7b'~ assay a ntenda0 to 0etarmne IAe amount d abc and n 0d
Principle TM GAnSals° assay nraswes ainc sad n oil n Ihs range d o m Io
ppm TAe meamd saes a proprrotary memprtutt raplwe rN 0vett arsry
d atrtc sad to tlpcWaphotomthc determnabon
rna r.nnaers- so tsanes pxsapaa vnm m Oro nagass an0 membrane unfit needed t0
run Ne assay The Y4 cancans ReageNA. Reopen 6,
EN6ort &dler. one Reagent Blank, arts Cahbramr Serape. and one Conbol Eadr 1ul
comes snlh 1M sppropnab rnon0er d dtaposabm
membnrn Milan and membrane urea
Sahfy Prsesubon: When used as dkacmd. the CanSah° roatpnts. cortnla.
and udbraton rhould prosent no harard to tb user Ar a
normal laboratory prscautrwt. avail contact wdh eyes and skm. Oa not Joel by
mouth
Production Packaoe: SaRyj'~1(orkatatian:plaoosabla
Three Bo<fb-Top Qspensen SalfesP"Labwaro.
Malyrar Kvmnpes
O~ Red Sa?esf~~ Fdbatmrt tJrul t3 mm Glue
Tesl Tubes
Heat 6104 Test Tube
Cape
Varba Ptpslte Tips
Poulne Displaewnanl RpHte
Adiurlabk Pipeee
t3 s 7 Smm Tent Tube
MuN Display Time
RBagetlU Dispenser Preparation NOTE &vrg rosgenn and dispenxrs W oom
tempanturo (te.x5'C) berara
uagwrvrg assay
t A6a an contaHS o! the roagent homes Dy gamy sw'aMp pram to attadmrg
dnpenxra.
2 AttaN the bspmrsn IabetM n aanql n'CMSa(e A' ono the nagaM bottle
IaOrJed'GOnSafe ~ Rasgent A' ant sat tM valumo to Us
spooled va4rtr m One GtnSa/e ~ assay nsfrtrdmn srarbon n the Sa?eat~' Bvrder.
7 Aeach the dnpan~r mbelsd n orange as'GtrtSde a' ordo Ne reagent bottta
40ded'CAiSa/e~~ Reagent 8 ' Dnpensa vdyme n
Ilaed. so no adprstmmt ~s necessary
1 Allach the dnpmner labeled n xanqe as'QmSafe Ehdmn BuIfIf onto the reagent
boele ta081ed'CdnSele~' Ebbon Bu2ef and xl Ns
voh~e b IM ipeafied vdwne m IM GmnSaft" pray rn»on:rrnwt h the SaITdt~' Ander
S Atladt ma dnpatser bbded m Isal ar'Prepuatrort Reagent onto au reagent boale
IaDascd'Prepsnhon Raagmt' Adpra the vakrme
scowdnp ro IM typo d t~ple bang tested Refer to ae Salfut" 6uWar far
Propsra6on Reagent volume
6 Oeparue approsunataly 1 to 5 dtquob al each napem ram a waste contauta to
shmnate any an W601as n IM Nspenses error to ura
SBMp(e Preparation Sa"pte peparatnn wn1 vary hued on IM pamad7r maNa matmil
Damp mrted Prepare andfaddute the samph appropnetely. accwdnq m
Pro samda proParatan seGwr n Ne Salrert° BuMa Be care m IoAOw all
Matug steps
Pre,Assay Preparation
t. Li0e1 one rort Wbe to Ne tbn6d are one test tube fp efdt 0d tamae to M mfmd
and p4Ce mfo Iha Salrefl"' Fdbatan Uml
t Label the yeaw C~M~rtd>ranar (OMU) anal 0t1 ton4ol and sample rrenben
1. Phce aro A4rttbnrsa Support Urof (MSU) wdh Ns Nbeled OMUs an He SaITasl~'
Fdtnbort Und. Uncap the 0lNs.
1 Uwp a poy6va QISpIacarKrN pipatta. bansler Ne mMrd and each oil sampb to the
caraapondtn9 OMU
S Recap th1 OMUs. cum an the S1?al"' FJfrabmt UnL and Mtv
t1 Far hydrogauNd ale. ptxs the of aamdes n an nwbalor ar Mst tdxk to 4puefy
pror m ddutron 1 2 Remove the MSU seepnp en
GMUs m place and remove Ute test NW s eon4uwlq tM Wlered Wa
7 Label a new sat of test brDas and detpnab arm aa'Waah'
6 Race the lNash 1' hrDes in IM SaITart° Fdtrslan UM. and roattadt me
MSU and OMUS Uncap me OMUs
9. U~g ate botUa~top dnpana, add EArtnn Buffer to eadr OMU and recap Tam on
the fa?est° Fdbatron Um! and filter pro samqes
After Hferofp n complete ant whm Ifu vacuum is aba on. rrnfudy unsaew each OMU
m rotaaal the vacuum arM gel Ne last drop d
halter vd0 the tube.
to. Rertwvs the AISU keep4p tM OMUr n Dlfce. Remove the lNash' prat tubas and
Place n a foal tube rock
24
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Test Method t. Label a new set or test epees r« tAa Reagent Blank. and
slaMarda The mMrol tubs and sampes for llYash t' tubes are atreedy
lanelaa
2 usvg an adluslabla ptpeW. dispense the reagera blank and salt standard mb
the dssqnata0 test tubes Refer b the GtnSahw
matrucbom m the SalTSSP' Baler to approprab vdurne for the roaqeM blank and
cahMa>ws
7 Dispense one rlqud d CbrSale° Reagent A uNO the reagent blank tube.
salt d the dose standard Nbes. Ua oontral tube. and salt
'Wash t-test tube
1 Dispense dro elquot d GtrrSale~' Reagent B rao the reagent maNr tube. each d
Me Itaee starbartl tubes. the contrd Wbe, and earn
'Wash 1'test tube
S Cap a9 the tuWs and voAw b IhorapMy rtes uP the solubarrs
8 Place as NWa (repent tdsnk sW lards. Qa~d. arH samples) mro a heat molt
Yortas the test tutxs hdae they qo mb the heat
mOtk, afld iqiM 111hen the tUbea C9Te 0W
7 Approsmately t0 msrutts before Me end d IM Aeatug penal. tum on the Sa?ess~~
Analyzer and allow d b urarm up Place Die
1301890 fdtsr m the Paler rpmpartmtM SderJ the -CITIPNR' program
d Mer the heabrp penal and foal rates, dank Ne Sa?set" Anahxer by eamtutq a
eda aontsrwg a rata monk losowe0 by t!r
Repent Bbnk h Uro Raapent BtsNt has an optxa! denaty (OD) roadutq greater than
0 O5. d rt unaocepta0le ant must be ropeated
9 CaAbate the nachme DY mserWg Standards as prompted by the Sanest" Mslyzer
tD. Foeoweg succaashd nh0rsbn. mM Iha omdrol tube TM cwttrd should be run evay
Ume ttro standu0s tm run To ensure IM
instrument and reapenls are perfonrdtp property. the conbd value should bC
wnDwt da renge staled on 9r paduga dfaen to IM
caratal pacsaged wah IM 4a (see Pacwa(p Insert ~ CarNal to CdnSaIe'~ O! Kds).
If the wrrtrd Wls srgrofiondy outcde the rargt.
prepare and run a hash conhal sampls
t 1 U W eon4d value lads vNAat 0ro range. urserl the sarnple tubes m Die
defrpnated enter
tt M the end d the day. stare standards. contrd. amt reagent bottles wdh
duperaas attached at room xmparaMe It 8 - 25rC)
Reporting Results t Tks Sanest analyzer woo use ma Wwatars b nlarute the owns
per rt~a, (ppm) of aax ado m od
3 II Ors sample va4e a groster than IM valve d Ilro hghesl stardaM. report a
n'~ (value of Mghssl standardl' For saampta. d the
hplxst standar0 value is 40 ppm and 01e umpk rosult at 50 ppm. flan ropoA d as
CIO ppm
1 Cltadv tM arstrvment prudout for Mps or srta mesasqes before reporonq
restate.
1 Far c led curve M (~ s 0 990). repeat the assay
The rungs M the canYd w bt,rtd on 9ro paeW qe insert pmwded wth Ins Cr~SaA~'
Od Control TM assay value rot tM control sfauld
approsrmate Ws range However. d a edmsea Nat sell tab esrebhsh a rbnboi range
usurp as own epmP~~ s~d persomel
LlrtlitBtlOTts The assay wdi pradva unra6s0h results for undutae ea<nplas a
penalty d~oWad samplss a run neat se you must duds and nn
AppllG2tiOf18 For spewl aPPhcataxa to Ws assay. eas ore SAI Product Support
Number t-88A.731.SAFE
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
APPENDIX B
Percent Fat Kit MSA for pet food, rendered meals, seeds, nuts and nut butter
PUfpo3e The Percera Faf assay is intended to determine the amount of lipid m
fat m a sample.
Principle
The Peg Faf assay measures ttre fat level in samples using enzymatic
hydrolysis of triglyeeraies to glycerol. followed by
an enrymatrclcolonmetric measurement of the glycerol released. The tnglycerrde
corxentratron is quanUtated m grams (g)
. per 100 grams of sample.
~ ne rarc~nr rar art comas pxaagea calm as the reagents and membrane amts
needed to nm Ute assay. The lul contains
Reagent A, one Calibrator Senes. and one Control. Each kit cortles rrrth the
appropnate number of disposable membrane
hol0ers and membrane ands The iut should be stored at 4'C.
Safety Preeautirn: Handle wNt care. The Puoerit Fat reagent carp stain
clothing and equipment When used es directed.
the Pant Faf reagents. conbols. and calibrators should present no hazard to
the Irsm. As a normal IaDoratorypreceuuon.
avod condo with eyes and skin. Do not o'roet by mouth.
Materials ReQuired
Producnon Pxkaoe: ;rafTesf~ Workstation: piaqwsabte labware:
One 8ottlaTap Dispensers Sa?saf~ Analyser Kunwipes
One Reagent Holder Ss?asf~ FJtration Unit 12 mm Glass Test Tutors
Heat Btoek Test Tube Caps
Vortex Pipette Tips
Positive Drsplacament Pipette
Adjustable Pipette
13 a 7 Smm Test Tube
Muter Display Tuner
SAI Heat Source (for Hydrogenated Oil
Samples)
Reagent/ Dispenser Preparation
NOTE. Bong reagents and dispensers to room temperahue (t&259C) before
beginning assay.
t Mix the contents of the reagent bonles by gently swirling prtor !o attatMrtg
dispensers.
2. Artaeh the dispenser laeded m red as 'Percent Fat A' onto the reagent
bottle IabNed 'Percarrf Fat A'. Dispenser
volume is Bxed, so no adustmml tt necessay.
3. Attach the dispenser labeled m teal as'Preparatan Reagent onto Me reagent
bottle labeled'Preparafron Reagent'
AOlust the volume according to the type of sampre being tested. Refer to the
Sa?dfr" Hinder for Preparation
Reagent vduma
t. Dispense apprprimately 4 to 5 aliquots d each roagent into a waste
container to eirmnate any air bubbles in the
dspensere poor to use.
Sample Preparation ~"ple ration wig va based on the ng repare andlor dilute
the sample
PWy particular matnx material bn tested. P
appropriately. according to the sample preperalan section in Ute
SalTist'°Oukk Start Card.
Pre-Assay Preparation
t. Label a new set of test tubes: ono for each M the eahbralors, RB for the
Reagent Blank, one tube for the control, and
t. 2. 3. etc for the samples. If sunning replicates, label the appropnate
amount of tubes la each control and sample
2. Using the adjustable ppette, Uansier the appropnate vdume of each
calibrator, reagent blank, control, and sample
side the designated test hrbes. Reler to the Percent Faf assay mstruetion
section m the SrITast"' Binder for
atpuoung volumes. Wipe Ne pipette tip poor to diSparsng all samples, reagent
blank, controls, and calibrators. Use
a new by for each sample, reagent blank. control, and calibrator.
NOTE: Run at least orta eonuol with each assay. The control will ensue that
the assay is being performed
correctly.
26
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Test Method
1. Dispense one aliquot al Percent Fat Reagent A into each of the eahbrator
tubes, the reagent blank, the control tube,
and each of the sample tubes.
2. Cap a9 the tubes and mveA gently to mix,
3. Place aN tubes fn the heat block set at 37-10°C for the time pencil
speafied m the Percent Fat assay mstruetwn
section found m the Sa?sat ~' Binder
6. Approxanately 5 mirnrtes before Ne end of the heating prowl Nm on the
Se?ea1~~ Anelyter aii0 aHOw it to warm up.
Place the SSa690 Blter in the filter cort~parSleet the'FATMASA' program.
5. Zero the instrument by inserting a tube contaimrrg a water dank fofbwed by
the Reagent Blank. b the Reagent 8lardc
has an optical densely (00) reader greater than 0.200, d is unaocep<abte and
must be repealed. Calibrate the
instrument by inserting the calibrators In the proper order as prompted by the
Sa?estr~ analyzer.
8. FoNovratg suaesstul ea4bradon. insert the wntrol tube. The contrd atauld
tra nm every time the catibratms are inn.
To ensure the tnswmem and reagents are perfartnmg propeAy. the control value
should fall within the range stated
on the package mse4 tar the bt d controls used (see Padeage lnseil - Contrd
for Peroent Fat IGts). tl 1he control
tails signrFCandy outsde the range, rerun the assay.
7. U the control value ta0s willnn the range, mseA the sample tubes m the
desamated order to analyze m the Sa?eat"r
Analyzer. Wipe each test lobe witA a Unt-free tissue prior to msertxxt m the
Sa?eat'"' Analyser.
8. At the end of the day, sloro eahbrators, contrd, and reagent bootee with
dispensers attached at room temperature
(2~°C).
Reporting Results
1. The Se?estr"' Analyzer w~U use the ealibraton: to ca~ulate the tnglyeeride
cancent2UOn m goons (g) of tngtycende
per t00 grams of oil.
2. U the sample value n greater dwn the value of the leghe~ calibrator. the
instrument inn Nag the result as'HL' The
sample must be diluted at a higher dihrUon and retested. Values that aro
Ragged 'Hr aro inaccurate and should rot
be reported.
3. Check the instrument pnnmut for (lags or error messages before reporting
results.
I. For tailed curve fd (~ < 0.990), repeat the assay.
5. The range for the contrd is found 0n the package insert provided with Use
Percent Fat cal Contrd The assay vefue
for Me control should approximate this range. The control result should be
multrplied by 400 to get the result
However. A iS advised !hat each lab establish a certroi rsrtgs using its own
!quipment and psrswinel
Llmitet)on! New food matrices widr no established protocols are considered
special appikalions and should be checked for
IMertwmce and spAie recovery. Refer to the Sample Preparation table found In
the Se?est° Binder.
Applications For coat
sp applications to Ihis assay, call the SAI Product SuppoA Number: i-888-321-
SAFE.
27
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
f'erc~frt ~f~~ rc~.~~ ~I~~~~rnrl(MSA) for Rendered meals, Pet Food
products, peanuts and peanut butter, seeds, cereals, and
encapsulated lipids
Introduction
The Safety Associates, Inc. Percent Fat Test Kit measures the fat level in
samples using an enzymatic hydrolysis
of triglycerides to glycerol, followed by an enzymaticJcolorimetric
measurement of the glycerol released. The
triglyceride concentration is quantitated in grams (g) per 100 grams of
sample.
Dispenser and Reagent Preparation
1. Mix the contents of the reagent A bottle by gently swirling prior to
attaching dispensers.
2. Attach the dispenser labeled "Percent Fat A" onto the reagent bottle
labeled 'Percent Fat A". The volume is
fixed at l.Oml.
3. Attach the dispenser labeled 'Preparation Reagent" onto the reagent bottle
labeled 'Preparation Reagent"
Dispenser volume is adjustable depending on the dilution for your products.
Generally 100u1 of the membrane
filtrate is combined with 2.4m1 of Preparation Reagent (Equals dilution of
1:100).
4. Dispense approximately 1 to 2 aliquots of Reagent A into a waste container
to eliminate any air bubbles in the
dispensers prior to use. To prime dispensers, please read the instructions in
the dispenser package.
Sample Preparation
Take your 1:4 filtrate and take 100u1 into a new tube and combine with 2.4m1
of
Preparation reagent . This is a 1:100 dilution..
Pre-Assay Preparation
1. Label a new set of test tubes: for the Reagent blank and five Calibrators;
one tube for the control; and 1, 2, 3,
etc. for the samples. Place 1 glass bead into each tube.
NOTE: If running replicates, label the appropriate amount of tubes for each
control and sample.
2. Using a positive displacement pipette or other pipetter, transfer 20 NI of
each calibrator, 20 NI of each control,
and 20 NI of each diluted sample into the designated test tubes. Wipe the
pipette tip prior to dispensing the
samples, controls, and calibrators. Use a new tip for each sample, control,
and calibrators.
NOTE: Run the control the first time a kit is used after opening and every
time the calibrators
are run. Subsequent sample runs without calibrators from the same kit on the
same day can
be run with or without controls depending on laboratory practice.
28
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Test Method
1. Tum on the SafTest"" Analyzer and allow it to warm up. Place the 5501690
filter in the filter compartment
and select the FAT1MSA program.
2. Dispense out 1-2 aliquots of Reagent A, into a waste container to ensure
that there are no air bubbles in the
nozzles.
3. Dispense one aliquot Percent Fat Reagent A into each of the calibrator,
control, and sample tubes.
4. Cap the tubes and gently invert 5 times. Place the tubes in a heat block
for 10 minutes (-37-42°C).
NOTE: After 5 minutes, perform a visual check to determine if samples will
fall within the range
of the curve. To perform a visual check, compare the color of the samples with
the color of
Calibrator 5. If the sample color is lighter than the color of Calibrator 5,
place the sample tubes
back on the heat block for the remaining time and proceed to Step 6. However,
if the sample
color is darker than the color of Calibrator 5, samples must then be further
diluted. First, complete
the assay for those samples that are lighter than the last calibrator. Then,
for those samples that
are darker than the last calibrator, begin at Step 5 for instructions for
further dilution and testing of
out of range samples.
5. If sample colors appeared darker than Calibrator 5 or if the SatTestT"
Analyzer gave a reading of "Hi at the
end of the assay, further dilutions may be required and are prepared as
follows: .
A. Prepare an additional 1:2 dilution on the sample already diluted for the
fat assay. (see sample preparation
section for percent fat assay) by adding 1 ml of the diluted filtrate to 1.0
ml of Preparation Reagent.
B. Label another set of glass tubes for the diluted samples and dispense 20 pl
of the diluted samples into the
appropriate tubes.
C. Dispense one aliquot of Percent Fat Reagent A into each sample tube, gently
invert and place for 20
minutes at 38-42°C in the heat block. After the mixing period, analyze
the samples using the STAT Mode
(see SatTestT"" Analyzer Instructions).
6. At the 'Blank Tube' prompt, insert a tube containing distilled water to
blank the instrument. Then insert the
reagent blank. Reagent Blank must have an optical density less than 0.20. Then
calibrate the analyzer by
inserting Calibrators 1 through 5 as prompted by the SatTestT" Analyzer.
7. Following successful calibration, insert the control making sure the values
of the controls fall within the
' ranges for the lot of controls used once multiplied by 400 for the dilution
(see Package Insert-Controls for
Percent Fat Kits). If the controls fall significantly outside the ranges,
rerun the assay.
8. If the control values fall within the ranges, insert the sample tubes and
analyze in the SatTest'"' Analyzer in
the designated order (see SafTestl"" Analyzer Instructions for detailed
instructions). Wipe each test tube
with a lint-free tissue prior to insertion in the SatTestT"" Analyzer.
Multiply your result by the dilution to get the
fat.
9. At the end of the day, store calibrators, control, and reagent bottles with
dispensers attached at refrigerated
temperatures of 2-8°C temperature.
10. To maximize lamp life, tum off the SatTestt"" Analyzer when not in use.
29
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Reporting Results
1. The SafTesf'"" Analyzer will use the calibrators to calculate the Percent
Fat content as percent fat n the
sample.
2. Adjust instrument results by taking into account the dilution factor:
Example:
Dilution SafTestT" ResultDilution x Final Result
Factor Result
1:80 0.32 0.32 x 80 25.6%
1:100 0.22 0.22 x 100 22%
3. If the sample value is greater than the value of the highest calibrator,
the instrument will flag the results as
'HL' Values that are flagged 'Hf are inaccurate and should not be reported.
Samples should be retested at a
higher dilution.
4. Check the instrument printout for flags or error messages before reporting
results. Erroneous flags may occur
for samples with values near zero.
5. Ranges for the Control are found on the package insert provided in the
Control Kit. Assay values for the
controls should approximate these ranges. However, it is advised that each lab
establish control ranges
using their own equipment and personnel.
NOTE:
If a STAT curve is going to be used, make sure that at least one control is
ran with each sample
batch and analysis. It is recommended that a new calibration curve be stored
weekly. Refer to
the SatTest'" Analyzer Instruction section for detailed instructions on sample
analysis using the
STAT mode.
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
~~v:~: _. ~~~:y .~.. : Kit STD (mayonnaise)
Purpose ~,e pint Fal assa is intended to detemdne the amount of 1
y ipid or fat m a sample
Principle The Percent Fat sass meawros the fat level m sam es usi en matte
drol sis of t
Y PI n9 zY M Y ~9~~des to glycerol, followed by
an eruymaUdcolonmetnc measurement of the glycerol roteased The tnglycende
concentratisori is quanhtated si grams (g)
per 100 grams of sample.
i nu rrrc~m rar xu comes Pacxageo warn as ine reegent9 arid membrane units
needed to run the assay. The tut contains
Reagent A, one Ca4brator Senes, and one Control Each tut comes with the
appropnate number of disposable membrane
holders and membrane unds The tut should De stored at ~'G.
Salafy tsreeautla~n: Handle wrath care The Percent Fat reagent wrap slaw
dothmg and equipment. When used es directed,
the Percent Fat reagents, controls, and cahtxators should present no hazard to
the user. As a normal laboratory preeaulron,
avdd ccnlaG vnth eyes and skin Do not oioet by mouth
Materials Required
Production Package: Sa?u!'v Workstation: Disoosabh Labwsn:
One tiot0e-Top Dispensers SalTesP" Analyzer Kimwipes
One Reagent Holder SeITesf"' Fdbatron Und ~ 2 mm Glass
Tesl Tubes
Heat Mack Test Tube Caps
Vortex Pipette Tips
Pomtrve Displacement Pipette
Adjustable Pipette
13 x 7 Smm Teal Tube
Mulk Display Tuner
SAI Heat Source (for Hydrogenated Od
Samples)
Reagent) Dispenser Preparation
NOTE: Bring reagents and dispensers to room temperaNre
(1B25C) before beginning assay.
t. Mix the contents of the reagent bottles by gently swuhng poor to attaching
dispensers.
2 Attach the dispenser labeled in red as 'Percent Fat A' onto the reagent
bottle labeled'Percenf Fat A' and set lha
vdume to the apeafied volume in lha Percent Fat assay insWCteon section in the
SatTastT~ Binder.
3. Attach the dispenser labeled in teal as'Preparauon Reagent' onto the
reagent bottle labeled'Preparatian Reagent.'
Adjust the volume according to the type of sample being tested. Rarer to the
SalfostT" Binder for Preparatxm
Reagent vdume.
4. Dispense approximately 1 to 5 a0quots of each reagent into a waste
con4arier to eliminate any a4 bubbles in the
dispensers prig to use.
Sample Preparation Sample preparation will vary based on the particular matnx
material being tested. Prepare aridlor dilute the sample
apDroprlately, according to the sample preparation sedan in the SaITest~'
Ouick Star) Card
Pre-Assay Preparation
1. label a new set of test tubes. one for each of the calibrators, R8 for the
Reagent Blank, one tube for the control, ar,d
t. 2, ~, ate. for the samples If running replicates, label the appropnale
amwnl of tubes for each tontrd and sample.
2. Using the adjustable pipette, booster the appropnate volume of each
calibrator, reagent blank, mntrol, and sample
into the designated test tubes Reler to the Percent Fat assay instruction
section in the Salfesf° Binder for
aliquohng vdumes Wipe the pipette hp prior to dispensuig a0 samples, reagent
blank, conVOls, and calibrators Use
a new by for each sample, reagent blank, contrd, and calibrator
NOTE Run at least ow control with each assay. The control will ensuro that the
assay is being periormad
correctly.
31
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Test Method
1. DYspense one aliquot of Percent Fef Reagent A into earn of the cahtxatw
tubes, the reagent blank, the control tube,
and each or the sample Wbes.
2 Cap all the tubes and rovert gently to mix.
3 Place aU tubes m the heat block set at 37-40'C for the bme perrod apea8ed m
the Percent Fat assay mstrudron
secron found ut the SalTest"' Amder
4. Approximately 5 minutes before the end of the mixing penod turn an the
SatfasF~ Malyzer and allow d ro warm up
Place the 5501690 nltar m the filter compartment Seled the'FAT/STD' program.
S. Zero the instrument by inserting a tube contamirg a water blank followed by
the Reagent Blank. U the Reagent Blank
has an opUcal density (OD) reader greater than 0 200, d rs unacceptable and
must be repeated Calibrate Ure
instrument by mserUng the calibrators m the proper order as prompted by the
SatTest~' analyzer.
8. Following successful rah6raUon, insert the control tube. The control should
be tun every time Ure cahbratore are inn.
To ensure the instrument and reagents are perfartning property, the control
value should fall within the range slated
on the package Insert for the Id of controls use0 (see Padrege Insert -
Control for Percent Faf IGIs). U the control
lags srgmficanUy wtsrde the rar>ga, rerun the assay
7. If the control value falls vwthin the renge, auerl the sample Urbes in the
desrgmated oMer to analyze in the SalTesf ~'
Analyzer. Wrpe each test tube wrath a Imt-free tissue prior to msertiwr m the
SaITestr~ Analyzer.
8. At the end of the day, store calibrators, control, and reagent Bottles
wrath dispensers aUached at room temperature
(2-8,C).
Reporting Results
t The SatTest ~' Analyzer will use lhr! calibrators fu calculate the
tngfyceride eoncentralron in grams (g) of triglycende
per t00 grems of od.
2 If the sample value is greater than the value of the highest cahbraror, the
msUument wdl Bag the result as 'HI: The
sample must be dduled at a higher dduhon and retested. Values that are Bagged
'HI' are inaxurete and shouts not
be reported.
3. Check the instrument printout for Bags or error messages before reporting
results.
4. For failed verve 6t (r: ~ 0 990), repeat the assay
5. The range far the control is found on tha package Insert provided wrath the
Percent Faf Od Control. The assay value
for the control should appronmale this range. The control result should be
mulbphed by 40D to gel the result
However, it is advised that each lab estabUah a canUOl range using ds own
eqmpment and personnel.
Limitations N~ f~ r"atMes mth no established protocols are considered speaal
apphrahom and should be drecked for
fnteAerence and spike recovery Refer to the Sample Preparalron table found in
the Saffesf~' Binder.
Applications For spewal appircatrons to 1Ns assay, caU the SAI Product Support
Number t-888-32t-SAFE.
32
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
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33
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
~r~8'.e~-~-s ~:
l~ercentFat(FATIMSA) Kit Quick Start Card
Matrix Kit
Dispenser and Reagent Preparation
~ Mix the contents of the reagent bottle by swirling prior to attaching
dispenser.
~ Attach the "Percent FaC dispenser labeled in red onto the Percent Fat
Reagent A bottle.
Dispenser Volume is set to 1.0 ml.
~ Attach the "Preparation Reagent" dispenser labeled in teal onto the
Preparation Reagent
bottle. Adjust volume according to the ~nrrl;~l: Pr~;mrr=~i~r~ C!ni~~;.~
~::lri G=Ir!~
To eliminate air bubbles, dispense approximately 2 to 3 aliquots of each
reagent into a
waste container prior to use.
Sample Preparation
~ Refer to the ~:mn;~l; i~l~fJ=lr_Itl'Jll ~!!ll:;: ~r:lrt G'-lrJfor sample
preparation instructions.
Pre-Assay Preparation
~ Label a new set of test tubes: Reagent Blank and C1 through CS for the
calibrators, and 1,
2, etc. for the samples.
~ Transfer 20 pl of each calibrator and 20 ~tl of the samples into the
designated tubes.
Test Procedure
~ Dispense one aliquot of Percent Fat Reagent A into each calibrator and
sample tube.
~ Cap tubes and invert 4 times. DO NOT VORTEX. Place tubes in a heat block for
10 minutes
at 40°C.
NOTE: If necessary, prepare an additional 1:2 dilution on the initial
filtrate. This will double
the sample dilution. For exampte, if the dilution of the initial filtrate was
1:100, the final dilu-
tion would then be 1:200.
Tum on the SaiTestT~" Analyzer and place the 550/690 filter in the filter
compartment.
~ Select the FATlMSA program and at the 'blank Tube' prompt, insert a water
black.
~ After 10 minutes, wipe tubes and insert the reagent blank and then
calibrators 1. through :5'.
as prompted by the SatTestT"" Analyzer.
Wipe and insert the sample tubes in the designated order for analysis and
results.
s ~ v~~~a~-~a~~:°~.~..c~c~~:.T~~ =,~.vr~~e~~ t~
34
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
APPENDIX C
STD Kit Assay Instructions
Standard Kit
Introduction
The Safety Associates, Inc. ProteSafer"'' STD assay measures the protein
concentration in oil samples as
milligram per deciliter (mgldl) or ppm (l0ppm is 1 mgidL)
Dispenser and Reagent Preparation
Allow reagents and dispensers to stand at room temperature (18-25°C)
for 15-20 minutes before beginning
the assay.
1. Mix the contents of the reagent bottles by gently swirling prior to
attaching dispensers.
2. Attach the dispenser labeled "ProteSafeT'" Elution Buffer onto the 500m1
reagent bottle labeled
"ProteSafeT''' Elution Buffer" and set the volume to 500p1.
3. Attach the dispenser labeled "ProteSafeT"" A' onto the 500 m) reagent
bottle labeled "ProteSafeT""
Reagent A". The volume is fixed at 1.Oml, so no adjustment is necessary.
4. Attach the dispenser labeled "ProteSafeT"' B' onto the 250m1 reagent bottle
labeled "ProteSafeT""
Reagent B.' The'volume is fixed at 150p1, so no adjustment is necessary.
S. Dispense each reagent into a waste container until there are no air bubbles
in the dispensers
(approximately 3-5 aliquots, slowly).
Sample Preparation
Make sure oil is homogeneous before sampling.
Pre-Assay Preparation
1. label a new set of test tubes: C1 through C4 for the calibrators, and 1,2,3
etc
for the samples. If running duplicates, label two test tubes for each sample.
Also, label the ely~ low ~-
Membrane Units (QMU) with the sample numbers.
2. Tum on the SafTestT" Analyzer and allow it to warm up.
3. Using the adjustable pipette, transfer 3001 of each calibrator into the
designated test tubes. Use a new
tip for each calibrator. Always wipe the pipette tip prior to dispensing the
calibrators.
4. Cap the test tubes to minimize evaporation and set aside in a test tube
rack until needed.
5. Set up the SatTestT'" Filtration Unit. Place the first six labeled test
tubes into the base. Attach a
membrane holder to the base, and then attach six-labeled D-Membrane Units
(QMU) to the membrane
holder. The C1MU labels should correspond to the labels on the test tubes.
Uncap the QMU's.
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Assay Performance
1. Prepare a 1:2 dilution of the oil sample by pipetting l.2ml of oil sample
into a labeled test tube. Set the
preparation reagent dispenser to l.2ml and add one aliquot of preparation
reagent to each oil sample.
Vortex gently and briefly to ensure homogeneous.
2. Using the positive displacement pipette, add 1ml of the diluted oil sample
to each QMU. Use a new tip
for each sample. Always wipe the pipette tip prior to dispensing the samples.
Recap the QMUs..
3. Tum on the SafTestT" Filtration Unit and filter the samples. Important.
Make sure the vacuum gauge
does not exceed 5psi on this step.
4. Repeat steps 2 and 3, total volume of 2ml filtered.
5. Remove the MSU with the QMUs in place. Remove the test tubes containing the
filtrates. Label new set
of test tubes and place them in the SatTestT" Filtration Unit. Re-attach the
MSU and ~MUs. Uncap the
QMUs.
6. Using the bottle-top dispenser, dispense 500NI of Elution Buffer to each
QMU. Turn on the SatTestT"'
Filtration Unit and filter the samples at 2-5psi. Tum off vacuum.
7. Repeat step 6. Do not change tubes.
8. ~temove the MSU with the QMUs. Remove the test tubes and place them in a
test tube rack.
9. Aliquot 300u1 of each sample from step 8 into a new set of labeled tubes.
10. Dispense one aliquot of ProteSafeT" Reagent A into each calibrator and
each sample tube.
11. Dispense one aliquot of ProteSafeT"" Reagent B into each calibrator and
sample tube.
12. Cap all of the tubes and INVERT GENTLY 3.d TIMES. Let tubes stand for 2
minutes then read.
13. Select'PROISTD' program in the SaiTestT" Analyzer. Place the 5TOI690
filter in the filter compartment
and blank the analyzer by inserting a water blank.
14. Insert Calibrators 1 through 4 as prompted by the SatTestT" Analyzer.
Insert the sample test tubes in
the designated order. Wipe each test tube with a tint-free tissue prior to
insertion in the SaffestT"
Analyzer.
Reporting Results
The SafTest'"'Analyzer will report results as milligram per deciliter (mg/dl)
of protein in the sample. This
can be converted to ppm by multiplying by 10.
2. If preliminary dilutions were made to the oil sample prior to testing, the
dilution factor must be taken into
account. Multiply the instrument results by the dilution factor to obtain the
value of the sample.
36
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
3. If the sample value is greater than the value of the highest calibrator;
the instrument will flag the results
as 'HL' The sample must be diluted to a higher dilution and retested.
4. If the sample value is less than the value of the lowest calibrator, the
instrument will flag the results as
'LO' and should be reported as '< (value of the lowest calibrator).'
5. Check the instrument printout for flags or error messages before reporting
results. A large coefficient of
variation (%CV) is expected with samples that are measured at the low end of
the calibration curve.
6. For failed curve fit (~ < 0.995), repeat the assay.
7. Ranges for the controls are found on the package insert provided in the
Control Kit. Assay values for the
controls should approximate these ranges. However, it is advised that each tab
establish control ranges
using their own equipment and personnel.
37
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Protein Content in Refined Oils Assay Instructions -
Introduction
The Safety Associates, Inc. ProteSafeTM assay is a rapid method that measures
the protein
content in an oil, tallow or grease by determining protein content captured on
a
chemically modified membrane which is then washed to release and concentrate
the
pmtein for analysis. Samples should be kept at 40°C when sampling,
placing on the
membrane and aliquoting the filtrate.
Dispenser and Reagent Preparation
Reagents and calibrators and controls are stored cold until used. The f rst
day of use allow
reagents to stand at room temperature (18-25°C) for 30-40 minutes
before beginning assay.
Once brought to room temperature leave at room temperature until all reagents
are used
except for calibrators and controls which are kept in the refrigerator.
1. Mix the contents of the reagent bottles by gently swirling prior to
attaching
dispensers.
2. Attach the dispenser labeled "ProteSafe A" onto the reagent bottle labeled
"ProteSafe
Reagent A." Dispenser volume is fixed, so no adjustment is necessary.
3. Attach the dispenser labeled as "ProteSafe B" onto the reagent bottle
labeled
"ProteSafe Reagent B." Dispenser volume is fixed, so no adjustment is
necessary.
4. Attach the dispenser labeled as "Prep Reagent" onto the reagent bottle
labeled
"Preparation Reagent". Dispenser volume is adjustable. To prepare a l2ml
sample,
set the dispenser at 2ml and use three aliquots. Combine with 6ml of the oil.
Warm
and vortex well.
5. Elution Buffer is added to the membrane in two SOOpI aliquots using a 1 ml
pipetter.
6. Dispense approximately 2 to 5 aliquots of each reagent into a waste
container to
eliminate any air bubbles in the dispensers prior to use.
Sample Preparation
Starting with heated oils make a 1:5 dilution using Preparation Reagent in a
15m1 conical
tube. Vortex the samples thoroughly. Place the diluted samples in a heat block
or water
bath for 10 minutes at 40°C. Sampling is critical to this assay. Remove
the samples from
heating source and vortex thoroughly. Apply Sml of diluted samples to a
membrane and
filter. Keep vacuum pressure less than 3 inches of Hg on the vacuum gauge.
Discard
filtrates and replace tubes with a new set of labeled tubes. Apply an addition
Sml of
diluted samples on same membrane and filter. Total volume of filtered sample
is l Oml.
Discard filtrates and replace tubes with a new set of labeled tubes. Using the
elution
buffer, wash the membranes two times with SOOuI aliquots. Collect the wash
filtrates far
analysis.
38
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Calibration Assay and Subsequent Testing
1. Label a new set of test tubes: C1 through CS for the 5 Calibrators; Control
for
the 0.0018% protein in oil depending on your application; and 1, 2, etc. for
the
samples.
2. NOTE: If running duplicate samples, label two tubes for the control and
samples.
3. Using a pipette, transfer 300 pl of each calibrator, control, and prepared
sample
into the designated test tubes. Wipe the pipette tip prior to dispensing the
samples,
controls, and calibrators. Use a new tip for each sample, control, and
calibrator.
4. Cap the tubes to minimize evaporation.
5. NOTE: Once calibrated run one control with each test.
Test Method.
1. Immediately tune on the SajTest''M Analyzer and allow it to warm up. Place
the
570/690 filter in the filter compartment and select the program PRO/HSY.
2. Dispense one aliquot each of "ProteSafe A" and "ProteSafe B" into each of
the
sample, calibrators, and control tubes.
3. Cap the tubes and gently invert 3-5 times. Place the tubes in a test tube
rack for a
period of 2 minutes at mom temperature (18-25°C).
4. At the 'Blank Tube' prompt, insert a tube containing distilled water to
blank the
instrument.
5. Calibrate the machine by inserting Calibrator 1 through 5 as prompted by
the
SaJTesfi'M Analyzer.
6. Following successful calibration, insert the control making sure the value
of the
control falls within the expected protein range for the lot of controls used
(see
Package Insert-Controls for Protein in Oil Kits). If the control is outside
the
expected value by 10%, rerun the assay.
7. If the control value is within the expected range, insert the sample tubes
and
analyze in the Saflest~'"~ Analyzer in the designated order (see Section 4 for
detailed instructions). Wipe each test tube with a lint-free tissue prior to
insertion
in the SafTesftM Analyzer.
8. Discard all used reagents.
39
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Reporting Results
1. The SaJTesf~'M Analyzer will use the calibrators to determine the protein
concentration of the control and samples.
2. Adjust instrument results by taking into account the dilution factor:
Example:
Saflesf~'M Result
Dilution Dilution x ResultFinal Result
Factor PPm
PPm
0.0018%
controlis
prepared 9ppm 2x9ppm l8ppm
at 1:2
and lOml
supplied
Sample
prepared 4ppm 2x4ppm 8ppm
the
same
3. Check the instrument printout for flags or error messages before reporting
results.
Erroneous flags may occur for samples with values near zero.
4. Mange for the Control is found on the package insert provided in the
Control Kit.
Assay values for the control should be within 10% of these values. However, it
is
advised that each lab establish control ranges using their own equipment and
personnel.
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Protein in Refined Oil Kit (PRO-HSY)
Purpose Tho p~~t SessonAeg assay is vrtended ro determure the artwuN of
seasoning on a clap.
Principle The Peroenl S meawrea the
easonurg assay protein level n seasanug on chips urrrtg a micrvlxotein assay
vnth oolprtnetrtc
endpoints. The protect concentration rs quantrtated ur miiGgrams (mg) per dL
and converted to % seasoning by comparison
ro a 696 eqwvalent seasonrrg conteol.
Kit Content9 The Percent Seasoning tut comes packaged catch all the roagents
and membrane uncle needed to tun the assay. The ke
contains Reagent A, Reagent A, packets ro preparo Preparation roagent, one
Calibraror Senes, and one Contrd. Each kd
comes with the appropriate number of dsposaDle membrane holder and membrane
units. The kit should bs stored at
4'C.untd used and left out aher fiat use for up to one monpr at roan
temperaturo.
Salary Pneautlon: HarMte wish taro. The Percent Seasoning wit wn9 stain
cbthing, hands, and equipment. W hm used as
directed, the reagents, canlrols. and calrbrarors should present no tra:ard ro
the user As a rwrtnal laborarory precaution,
avord contact wrath eyes and skin and wash copously il on skin or m eyes. Do
not bloat by mouth. Do not ingest, as ors
component a roxrc
Materials Requited
PraducYron Paekaa~; S3lTesi~~ Workstation: Diaoosable Labware:
Two Bodle-Tap Dispensers ~Iresf~ Analyzer Kimwqres
Orro Reagera Holder SafTesf" Fduatrart Umt 12 mm Glass Test
Tutus
Test Tree Caps
A~r~ble Pipette Piperte Tips
13 : 7 5mm Test Tubes 1 Op~'t ~~ ~ stn
Mule Orsptay Tuna Plastic baggres
Reagent! Dispenser Preparation
NOTE: f3nnp roagents and dispenser tn room temperaturo (1a-25'C) befom
beginning assay.
1. Mx the contents of the roagent bottles by gently swirling prior ro
attactung dopmsers.
2. ArtacA the d'rspenser labeled 'Seasoning A' onto the roagent Dotde labeled
"Percent Seasoning Reagent A' and
for "Seasoning 8' onro the rosgent bottle labded'Percent Seasorunp Reagent 8.
The volumes era preset to the
sperdred volumes m the Percent Seasoning instruction seebon in the Sa/fist"'~
Binder.
3 To proparo Preparation Reagent add the contents of ors packet
ma*ed'Proparation Reagent Salts' mro SVters
of distilled water.
4. Mix for 5 minutes using a sbr bar ur a large beaker. Save for use in assays
drrpughout the day.
5. Dispense approrumately 1 ro 2 aliquots al roagents A and B mro a waste
container to eliminate any air bubbles in
the dispensers prior ro use.
Sample Preparation Sample preparotron vnU vary based on the particular
seasoning trorng lasted. Propane arrNor dikrte the dnp sample
appropnately, acoordng ro the sample prepa2tion section ur the SaJTesNr Owck
Start Card.
Pre-Assay Preparation
1. At the Arst assay each day, label a new set of test tubes: one fw each of
the cahbrarors, one tube for Ne control,
and 1. 2, 3. etc. for the samples. tl runrting repGCates. label the eppropnate
amount d tubes for each control and
sample.
2. Usug the adjustade pipette. Uansler the appropnale volume o1 each
calibrator, control, and sample into the
deagnated test tubes. Refer ro the Percent Seasoning instruction section m the
Salfest~- Binder Iw aliquoting
volumes Wipe the pipette tip poor to dispensing all samples, oonuols. and
eahtuarors. Use a new by for each
sample, reagent blank, control. and cai~ratw.
3. After the hrst cahbrahon Assay, use die STAT mode and ram one control and
one chip sample each lime point.
NOTE. Run at least ow control with each sample. TM control will ensuro that
the assay is being performed
correctly and that the Chip sample is within expected % seasoning range.
41
SUBSTITUTE SHEET (RULE 26)
CA 02470724 2004-06-16
Test Method 1. Fw the Brat assay al the day loin on the SeITest'" Analyzer tw
5 minutes Plate the 5701690 litter yr the hltm
compartment. Seleetthe'SEA/FL'program.
2. Dispense ono aliquot of Percent SNSOVrting Reagent A into each al the
cakbrator Nbes, the cantrd tube, and each
01 the sample tubes. Dispense one afiquat of Portent Seasoning Reagent 8 unto
each of the calibrator ties, Ore
control tube. and each of the sample tubes.
3. Cap as Ure drbes and 'amen gendy 3-5 times to rtns.
4. Set lima for 2 mrmrtea. For the first assay loin on dro Sal7aa~~ Analyser
for 5 minutes. Place Ure 57N690 finer rn
rite form compartment Seled the "SEAIFI.' program.
5. Zero the instrument by irrserdng a tube contarrkrg warm blank. Calibrate
the hrstnm>da by mxrting the
eakbrators n Hte proper ordm as pranpted by the SalTesr~r analyzer
6. FaUowutg successful cel~bralron, insert the control tube. The control
should be rim every bme the samples ara run.
To enauro the mstrumem and reagents are pertortnirg properly. the coned value
should fall wilier Ne 2nge
stated on the package insert for the lot of controls used (see Package Insert -
Control for Pertelrt Seasoning Krts).
N the eerard fa0s signilkantly autsde the range, renrn the assay.
7. d the control value 411s within the range, utsert the sample tubes in the
desgnaled ordd to analyze m the
SaITear~" Analycm. Wipe each test Nbe vnth a dnt~free tissue prior to
insertion m the Ss?eat~' Analyzer.
8. At the end of tha day, store cafbrators, control m the refrgerata. artd
reagent bolUes vrnth dispensers attached at
roam temperature '
Reporting Results
1. The SalTist'~ Analyzer wr9 use the calibrator to cakvlate the protein
oontentrseon m mgrdL of controls and
the sdubJized ehro.
2. If Ne sample value is groater 0ran the value of the higheu cakbratar. tM
instrumem wrB lfag Ore resuh as 'HL'
The sample mud be tfiluted at a hghm dilution and rctested. Values that are
(lagged 'Hr are rrraowrate and
should not be reported.
3. Check the mslnunenl Printout for bags or error messages beforo reporting
results.
1. Fw laded tune fit (t < 0.990), repeat the assay.
5. The rage for tM control is found on Ure package 'orsert provided with the
Parfait Seasoning Condd. Ths
assay value for pre control should approximate this range.
LlmitaNons New food matrices vnth no established protxds aro considered apedal
applications and should be checked for
interferenea and spike recovery Refer ro the Sample Preparation table lound m
the Silfsst"' 8mder.
Applications poi spedal appkcatrons to 0ris assay, can the SAI Product Support
Number. t~BB8-321-SAFE.
42
SUBSTITUTE SHEET (RULE 26)
