AU2002365053A1 - Devices and methods for isolating and detecting specific substances in complex matrices - Google Patents

Description

DEVICES AND METHODS FOR ISOLATING AND DETECTING SPECIFIC SUBSTANCES IN COMPLEX MATRICES Related Applications 5 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 10 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. 20 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 25 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 30 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 1 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 5 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 10 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 15 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 20 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 25 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.). 30 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 2 means of determining the minimum amount(s) 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 5 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 10 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. 20 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 and/or 25 concentrations of certain analytes or the presence of certain nucleic acid sequences in complex matrices, so that relatively untrained -personnel may perform 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. 3 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, 5 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, 10 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 15 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 20 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 25 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 30 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 4 collection of the filtrate(s) 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. 5 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 10 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 15 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 20 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 25 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 30 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 5 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 acid(s) from the sample prior to analysis for the FFA. To accomplish this, the sample is passed 5 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 10 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 15 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 20 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 25 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 30 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 6 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 5 substance or a protein, that may be present in the sample. The bound nucleic acid(s), genetically modified substance(s) or protein(s) 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 10 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 20 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 25 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 30 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. 7 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. 5 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 10 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 20 the Saftest

T

M Membrane Unit and the SaftestTM 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 25 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 30 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 8 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 5 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 lo embodiments, the samplel8 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 15 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 20 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 25 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 30 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 9 have been used to capture another analyte(s), such other analyte(s) may be eluted from the membrane(s) 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 5 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 o10 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 15 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 20 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 25 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 30 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 10 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 5 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. 10 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 15 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 20 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 25 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. 30 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. 11 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. 5 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 0lo protein binding membrane by passing 1 to 200ml of oil through the membrane and eluting the protein off into another tube using a salt solution in I 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. 20 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. 25 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. 30 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 12 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. 5 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 10 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 20 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. 25 Detailed Examples Of Specific Embodiments Of The Present Invention 30 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 13 in commonly owned PCT International Patent Publication No. WO 99/20396 and U.S. Patent No. 6,489,132, and the publicly available SafTestTM 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 5 reference. Example 1 Separation and Determination of Free Fatty Acid (FFA) and Citric Acid in an Oil Sample 10 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 15 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 20 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 25 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 30 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). 14 The CitriSafeTM 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 5 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 FASafe T M from Saftest, Inc. (Phoenix, AZ). The CitriSafeTM and FASafeTM test kits are useable in conjunction with devices described in copending United States Patent 10 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 Saftestr m Filtration Unit or SaftestTM Work Station, from 15 Saftest, Inc. Example 2 Removal of Interfering Inorganic Acids and Determination of Free Fatty Acid (FFA) and Acetic Acid in a Food Sample 20 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. 25 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 30 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 15 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. 5 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 pi 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 10 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 FASafeTM publicly available from Saftest, 20 Inc. (Phoenix, AZ). The FASafeTM 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 SaftestTM Membrane Unit and the SaftestTM Filtration Unit or SaftestTM Work Station, from Saftest, Inc. 30 16 Example 3 Determination of Total Fat Content and/or Percent Fat This example demonstrates the determination of fat content or 5 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 10 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 pL) 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. 20 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 30 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 17 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 SaftestTM Filtration Unit or SaftestTM 5 Work Station, from Saftest, Inc. Example 4 Determination of Protein Content in Refined Oil 10 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 20 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 0C). The presence of protein is 25 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 30 as ProteSafeTM from Saftest, Inc. (Phoenix, AZ) and is described in Appendix C to this patent application. The ProteSafeTM test kit is useable in conjunction with devices described in copending United 18 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 5 SaftestTM Membrane Unit and the SaftestTM Filtration Unit or SaftestTM Work Station, from Saftest, Inc. Example 5 Determining the Presence Of A Particular Microbe in a Food Sample 10 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 15 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 20 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 25 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 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 5 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 rd Edition, 2001. The PCR products are labeled 10 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 15 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 20 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 25 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. 20 APPENDIX A CoitriSfe - STD Kit Assay Instructions Standard Kit Introduction The Safety Associates, Inc. CitiSafe

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m 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 (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 in yellow as "CitriSafe Elution Buffer" onto the 500 ml reagent bottle labeled "CitriSafe Elution Buffer" and set the volume to 750 pl. 3. Attach the dispenser labeled in yellow as "CitrlSafe 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 ml 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 pl of the reagent blank and 750 lil 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 4. Set up the SafTestTM 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 (QMU) 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 sarmnple. Always wipe the pipette tip prior to dispensing the samples. Recap the QMUs. 2. If testing hydrogenated samples, warm them until liquid and dilute 1:2. 3. Once the sample is diluted analyze as usual. 4. Turn on the SafTestT M 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 SafTest T M Filtration Unit. Reattach the MSU and QMUs. Uncap the QMUs. 6. Using the bottle-top dispenser, dispense 750 pl of Elution Buffer to each QMU. Turn on the SafTestm Filtration Unit and filter the samples at 1-2 psi. 7. After filtering 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 (100pl) Reagent B into the reagent blank tube, each of the three standard tubes, and each sample tube. 11. 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 SafTestM Analyzer and allow it to warm up. Place the 4201690 filter in the filter compartment. Select "CITISTD" program. 13. After 30 the minute heating period, blank the Safrest

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m 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 SafTest

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m 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 SafTestT Analyzer. 22 Reporting Results 1. The SafTestP'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 (%CV) is expected with samples that are measured at the low end of the calibration curve. 6. For failed curve fit (r2 < 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 Kit for Oils Purpose The CiNiSsfe~ assas intended to determine the. amount of tite " d Cd Principle m .tee esa esesOrCms o i weofO40pmThu methond uses a poprielany m mbrecapttjre ad dna assay d MK Wiad for ipcrpwntiideterminatin Kit Contents The Cdiid t ulcoet pactiaged wit ad the mreeetson M teratiewiltietnsd ft s ssaY The kit ftatitReagertA. RsageS . Etltet Suffer, cOte Reegutit Btankti arm, calibrator Series. wid amS Commle Each kit Comes with the Appropite tMaribor of despo~s menibran. holers andl mterbrnits Safet Precaution: When used as directed. the C.*tSaionr reagetnts. coniusts. arid cetraterl shouldessr enlto aWord 10Pr er As a minttel laborator, Precaution. avoid contaat with eye& and Afsokauc lt ym Materials Required PrsoWIimasPaage' 91socisable Latrwars Three Bttle-Top Ottoetier Saffest.. Midyear t(Amtwives One Reagent Holder Seffost- FdrmUi 12 min Gluts Test Tuas. Heat Blck ~ Test Tube Cape vortex Pipette Ttle positive Ouiptacemneut Pipette Adiuutable Ptteu 13? a mm Test Tube Mutt Display Titte Reagentil Dispenser Preparation NoTe anng agents, and dipenisers to rett temperature lie-25C) before begunt"i assay I Aie the contests of the reagent hewtt by getly uiviring over to attachig ispensers. 2 Allach the dispenser labeled in orange as 'CitrtSae A& aft ft reagent boblaeleed Ct,,Sefe Reagent A awi set tvournts to ilts epeoifed vicoume i the Cotrt$ale assayinstruction - in the Safrosat Sier. 3 Attach the dispenser tlabeled i orange as Cittutee W' otto ft reagent Pa b otteleltd iisafe"- Reagent e0 Dispense, sctwuu is Seed. so no adjtmient a necessary 4 Attach the dispenser labeled in orange as OC~tiafe Eluton Snufer' onte the reagent bolse labeled CleSalem Ekittn Butte and set Reo volumee to thle specified volumesan tie CtiSaele' essay intruction Section in the Saefl' Bider 5 Atach the dispernsrtlabeled in test os 'Pheperebot, ReegeW sonts tie reaget ets tabeled -Prepaatot. Reagent' Adpri Re volume accordig to thN type of earette being tested ReON lt e Ssfrest' Smiler tsr preparaton Reagent nolume a Dispense approsernalefy 4 toO etaiquots of eacti reagentl into a waste cents~n to etoltatete any air boubblems Rnoe dispeanar picr to ut. Sample Preparation Samoatepreparation ettvery based sit tOe parltclar metrit materalbeing tased Proep ngar idtule Resample eptpnete 1 . ardeig to Si. saitelepreparaton seton mfeSaiMest'BinderBewet soiaheinsep Pro-Assay Preparation 1. Law or lstuefre Coto an amc test tube for each art sample to be tested and Place into Rhe SafrlmJ Filration Unit 2. Label the roguw 0-Piemrbravies IGMIJ with te control and sampte imtiers 2. Place Ons Membtte Support Unit (USU) with the labeled 0MUs on SUe Safrost"' Filtain Unit. Uncep the OUS 4 Using a posii ilaeenrt p~pette. transfer the con his and eacadt sample tofte corespoing OMtJ 5 Recap the OMUe. tm oi te Saffeet'a Pitraeon Utet. and fier O Per hydrogenated ads. place the ad eame i m icubator of hoet block to iquefy prior to dilution 12 Remove the MttJ keeping Rhe OUs mv ptaise end trsins ft test Noes Comnog the tittee ads ? Label a new set ol lest tbatts and designed Rhem atsWi, 8 Place the 'Wesh V tie In Re Sarr'eat' Fltebn Uret. and reattachi tie MSU and ONUs Uncap the OAtUs 9. Usin the bottle-top, dispener. add Eltett Bufter to amA olU and recap Turn Oni the Saneostt Filtration it anIldrttohe samples Aftter Weing is complete arnd wide Re vacuum is stitl sit. care"t unscrew each OMU to release the viacuaum anid got he last drop of buffer ilto Rhe tube. tO. Remtore Rea MSU keepding thes Otas n place. Remove Re 'Wail? test tubes Arnd Place elts test hbte rack 24 Test Method I. Label a new set of mest tuibes for t Reagent Btank, and Standards The constr tube and samples tor We r I ts ubns ere basiy labeled 2 Using an astijuslabe p petle. disperse the reagenst blank and each standard into the designated ast tubes Refer to re CfrnSlem instructions n the Safrsf* Bandr tr p piopraale volu M for the reagent blank and cbbators 3 Dispense one atquot of CdnSafte Reagent A sinto the reagent blarnk tube, each of the three slandanj tubes, the control ube. an ech Wash 1 tet ube 4 Dspense one hqut of CdnSafew Reagent B a the reagent blank tube, each of the three sandard tubes. the control tube, and each "Wash l' tenst tube 5 Cap adl the tubs and vsertes It thoroughly mi. up the solubans 6 Place all tubes (reagent blank standards. conto. and samplesi rin a heat black Vatx the tent tubes before they go to the heat block. and again when the tubes ome A 7 Approxoiately 10 rminuts before the end of the heatmn Denad, afn On the Safres t M Analyzer and allow a to warm up Place he 420690 Ilter a the liler cmiettet Siet the 'CITIPNF program 8 Ater the heating pered and S nal werex, blank the Saffest" Analyzer by Minserting a tube containing a wate blank allowed by the Reagent Blank If the Reagent Blanit has an optical density (O0) reading greater than 0 05. d as unacceplable and musl be repealed 9 Calibrate the machine by msa ting Slandards as prompted by he SafTest' Analyzer 10. Following successful calbbihn ssernst the central tube The Control should be run eveY irtme e slandarls ae urtn To ensure the instrument and reagents are perfomng property. the central value should fal wtlun the range stated on *ii package isert for the central packaged wrilh this kt (see Package Insert. Carin for It n riSafe " Oi Kits). If he control fails 5nicarly outde the range,. prepare and run a fresh ontral sample $1 Ifthe control value lals waihn the range. mnet the samplpe tlruibes in te desgnated order 12 At the end of the day. store slandardei. conhol, and reagent bottles wih dispensers attached at roon temperature(18 - 2VC) Reporting Results I The Slarrest m Analyzer wila usa the cabburators ctalcutat the parts per runo (ppm) al afc acid ina 2 If the sample valie is greater than the value of the highest standard. report it as "& (value of highest standard) * For ample. If the h Highest standard vah1 is 40 ppm and the sample result s 50 ppm. than report d as 40 ppm 3 Check the manbrumesner printout for lags or error messages befotare report re sultt . 4 For faled curve fi ( - 0 990). repeat the assay 5 The range fe the control laund on Ore package rosart provided with me C ,rSalem d Control rThe assay value for thiseotrol shoud approsmale S tis range However. do adved Is eac lab estMabhs l a conitrog range using s own e Ipelllpnt aM d personnel Limitations The assay w produce unremable results for insoluble samples or partially dissolved samples if runm neat so you must dduAle and et Applications For spe"a appcations to oas assay, cal Oe SAl Produci Support Number 1-888-321SAFE 25 APPENDIX B Percent FatKit MSA for pet food, rendered meals, seeds, nuts and nut butter Purpose Purpose The Percent Fat assay is intended to determine the amount of lIpid or fat in a sample. Principle The Percent Fat assay measures the fat level in samples using enzymatic hydrolysis o triglycerides to glycerol. followed by an enzymatiCcolonmetric measurement of the glycerol released. The tnriglycende Concentration is quantitated a grams (g) per IOU grams of sample. Kit Contents The Percent Fat lokit comes packaged with all the reagents and membrane units needed to run the assay. The kul contains Reagent A. one Calibraltor Senries. and one Control. Each kit comes with the appropriate number of disposable membrane holders and membrane units. The kit should be stored at 41C. SafetyPrecaution: Handle wh care. The Percent Fat reamgent wiv stain clothing and equipmenL When used as directed, the Percent Fat reagents. controls. and calibrators should present no hazard to the user. As a normal laboratoryprecaution, avoid contact with eyes and skin.o o nt tb mo . Materials Required Production Paikage: SafTesf" Workstation: Dieoable Labware: One Bottle-Top Dispensers Saf'est" Analyzer Kurwies One Reagent Holder Sa ges" Fdiltration Unit 12 minm Glass Test Tubes Heat Block Test Tube Caps Vortex Pipette Tips Positive Displacement Pipette Adjustable Pipettle 13 x 7 5mm Test Tube Multi Display Timer SAl Heat Source (for Hydrogenated Oil Samples) Reagent/ Dispenser Preparation NOTE. Bnng reagents and dispensers to room temperature (18-259C) before beginning assay. 1 MIx the contents of the reagent bottles by gently swilling prior to ataching dispensers. 2. Attach the dispenser labeled a red as "Percent Fat A* onto the reagent bottle labeled "Pecent Fat A'. D spenser volume is fixed. so no adjustmentl is necessary. 3. Attach the dispenser labeled in teal as "Preparation Reagent onto the reagent bottle labeled "Preparation Reagent" Adjust the volume according to the type of sample being tested. Refer to the Saffestr Binder for Preparation Reagent volume. 4. Dispense Approximately 4 to10 5 aliqkuotls of each reagent into a waste contuner to eirmnate , ay air bubbles in the dispensers pnoor to use. Sample Preparation Sample preparation will vary based on the particular matrix material being tested. Prepare andlor dilute the sample appropriately, according to the sample preparation section in the Saffest Quick Stanrt Card. Pre-Assay Preparation 1. Label a new set of lest tubes: one for each of the calibrators. RB for the Reagent Blank, one tube for the control. and 1, 2. 3. etc for the samples. I runnmig replicates, label the appropnate amount of tubes for each control and sample 2. Usming the adjustable pipette, transfer the appropriate volume of each calibrator, reagent blank, control, and sample into the designated test tubes. Refer to the Percent Fat assay instruction section in the SafTest' Binder for ahiquoting volumes. Wipe the pipette tip prior to dispensing all samples, reagent blank. controls. anrid calibrators. Use a new lip for each sample, reagent blank. Control. and caliber tor. NOTE: Run at least one control with each assay The control will ensure that the assay is being performed correctly. 26 Test Method I. Dispense one aliquol of Percent Fat Reagent A into each of the calibrator tubes. the reagent blank, the control tube, and each of the sample tubes. 2. Cap all the tubes and swvert gently to mix. 3. Place all tubes in the heat block set at 37-40gC for the time penod spoafled an the Percent Fat assay instructon section ond in the SaffTeat Smder 4. Apprommately S minutes before the end of the heating period turn on the Saftrls'F Analyzer and allow it to warm up. Place the S=ESD filter in the filter compartman Select tthe "FAT/MSA program. 5. Zero he Instnrument by msarthng a tube contalnig a water blank followed by the Reagent Blank. tr the Reagent Blank has an optical density (00) reader greater than 0.200. it is unacceptable and must be repeated. Calibrate the instrument by inserting the calibrators In the proper order as prompted by the Saffest analyzer. 6. Following successful calibration, insert the control tube. The control should be run every time the calibrators are run. To ensure the Instruimen and reagents are pedorming property, the control value should fall within the range staled on thie package Ose for thes lot of controls used (see Package Insert - Contrat for Percent Fat IKts). It the control falls significantly outside the range, renin the assay. 7. II the control value fags within the range, rinsed the sample tubes in the designated order to analyze in the SafTest" Analyzer. Wipe each test tube with a lint-free tissue prior to assertion in the SaffeTeat' Analyzer. 8. At the end of the day. store calibrators. control, and reagent bottles with dispensers attached at room temperature (2-6~). Reporting Results I. The SafTest' Analyzer will use the calibrators to calculate these tnglyceride concentration in grams (g) of triglycerode per 100 grams s oad. 2. If the sample value is greater than the value of the aghliest calbrator, the instrument will flag the result as 'HI.' The sample must be diluted at a higher diluton and retested. Values that are flagged 'Hr are inaccurate and should notl be reported. 3. Check the instrunsment pnntout for flags or error messages before reporting results. 4. For adied curve fit (r' - 0.990). repeat the assay. 5. The range for the control s found on the package insert provided with the Pacent Fat Od Control The assay value for the control should approarmale this range The control result should be multiplied by 400 to get the result. However, it Is advised that each lab establish a control range usi5g its ow equipment and pjrsonrinel Umiritatlons New food maltices with no established protocols are considered special applications and should be checked for ntedlrence and spoke recovery. Rtefer to the Sample Preparation table found In the Safrest Binder. Applications For special applications to this assay, call the SAl Product Support Number t-888-321-SAFE. 27 Percent Fat Te.st hot/1d(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 enzymatic/colorimetric 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 1.0ml. 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 1 00ul of the membrane filtrate is combined with 2.4m) 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 100ul into a new tube and combine with 2.4ml 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 pl of each calibrator, 20 pi of each control, and 20 pl 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 Test Method 1. Turn on the Satlest

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M Analyzer and allow it to warm up. Place the 5501690 filter in the filter compartment and select the FATIMSA 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 0 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 SafTestTO 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 SafTestTM 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 SafTest T m 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 rail significantly outside the ranges, rerun the assay. 8. If the control values fall within the ranges, insert the sample tubes and analyze in the SafTest

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M Analyzer in the designated order (see SafTestr T Analyzer Instructions for detailed instructions). Wipe each test tube with a lint-free tissue prior to insertion in the SafTest T M 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, turn off the Saffest M Analyzer when not in use. 29 Reporting Results 1. The SafTestTm Analyzer will use the calibrators to calculate the Percent Fat content as percent fat in the sample. 2. Adjust instrument results by taking into account the dilution factor. Example: Dilution Factor SafTest T m Result Dilution x Result Final 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 'HI.' Values that are flagged 'HI' 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 SafTestM Analyzer Instruction section for detailed instructions on sample analysis using the STAT mode. 30 P:,.. r ZKit STD (mayonnaise) Purpose Purpose The Percent Fat assay is intended to determine the amount of Ipid or fat in a sample Principle The Percent Fat assay measures Ihe fat level in samples using enzymatic hydrolysis of triglycendes to glycerol, followed by an enzymatlc/icolonmetnc measurement of the glycerol released The tnglyceride concentration is quanilitated in grams (g) per 100 grams of sample. Kit Contents The Percent Fat kit comes packaged with all mthe reagents and membrane units needed to tun the assay. The kst contains Reagent A. one Calibrator Senes. and one Control Each lkit comes with ithe appropriate number of disposable membrane holders and membrane units The kut should be stored at 4C. Safety Precaution: Handle with care The Percent Fat reagent wAll stain clothing and equipment. When used as directed the Percent Fat reagents, controls, and calibrators should present no hazard to the user. As a normal laboratory precaution, avoid contact with eyes and skin Do not oet by mouth Materials Required Production Package: Safest" Workstation: Disposable Labware: One Bottle-Top Dispensers SafTesf" Analyzer Kimmpes One Reagent Holder Satesa" Fdtration Unit 12 mm Glass Test Tubes Heat Block Test Tube Caps Vortex Pipette Tips Positive Displacement Pipette Adjustable Pipette 13 x 7 5mrn Test Tube Multi Display Timer SAl Heat Source (for Hydrogenated Oil Samples) Reagentl Oispenser Preparation NOTE: Bring reagenls and dispensers to room temperature (18-.25C) before beginning assay. 1. Mix the contents of the reagent bottles by gently swirhling prior to attaching dispensers. 2 Attach the dispenser labeled in red as 'Percent Fat A" onto the reagent bottle labeled "Percent Fat A' and set the volume to the specfled volume in the Percent Fat assay instruction section in the SafTest'" Binder. 3. Attach the dispenser labeled in teal as "Preparation Reagent' onto the reagent battle labeled "Preparation Reagent.' Adjust the volume according to the type of sample being tested. Rarefer to the Saffest'" Binder for Preparation Reagent volume. 4. Dispense approximately 4 to 5 aliquots of each reagent into a waste container to eliminate any air bubbles in the dispensers prior to use. Sample Preparation Sample preparation will vary based on the particular matrix material being tested. Prepare andfor dilute the sample appropriately, according to the sample preparation section in the SafetTesat" Quick Start Card Pre-Assay Preparation 1. Label a new set of test tubes, one for each of the calibrators, RB for the Reagent Blank, one tube for the control. and 1.2, 3. etc. for the samples If running replicates, label the appropriate amount of tubes for each control and sample. 2. Using the adjustable pipette, transfer the appropriate volume of each calibrator, reagent blank, control, and sample into the designated test tubes Refer to the Percent Fat assay instruction section in the Saffest " Binder for aliquoting volumes Wlpe the pipette tip prior to dispensing al samples, reagent blank, controls, and calbhlrators Use a new lip for each sample, reagent blank, control, and calibrator NOTE Run at least one control with each assay. The control will ensure that the assay is being performed correctly. 31 Test Method 1. Dispense one aliquot of Percent Fat Reagent A into each of the calibrator tubes, the reagent blank, the control tube. and each of the sample tubes. 2 Cap all the tubes and invert gently to mix. 3 Place all tubes in the heat block set at 37-40"C for the time period speckled in the Percent Fat assay instruction section found in the Saffes t s Binder 4. Approximately 5 minutes before the end of the mixing penod. turn on the Saffest' Analyzer and allow I to warm up Place the 5501690 filter min the filter compartment Select the "FAT/STD" program. 5. Zero the Instrument by inserting a tube containing a water blank followed by the Reagent Blank. If the Reagent Blank has an optical density (OD) reader greater than 0 200, it is unacceptable and must be repealed Cahlibrate the instrument by inserting the calibrators in the proper order as prompled by the SafTest' analyzer. 6. Following successful calibration, insert the control tube. The control should be run every time the cabrators are run. To ensure the instrument and reagents are performinng property. the control value should fall within the range stated on the package Insert for the lot of controls used (see Package Insert - Control for Percent Fat Kits). If the control fats significantly outside the range, rerun the assay 7. If the control value falls within the range, nsert the sample tubes in the designated order to analyze in the SaTffes t s Analyzer. Wipe each test tube with a lint-free tissue prior to insertion in the SaflTest T Analyzer. 8. At the end of the day, store calibrators, control, and reagent bottles with dispensers attached at room temperature (2-6*C). Reporting Results 1 The SaflTest m Analyzer will use the calibrators to calculate the triglyceride concentration in grams (g) at triglycende per 100 grams of oil. 2 If the sample value is greater than the value of the highest calibrator, the instrument wil flag the result as 'HI.' The sample must be diluted at a higher dilution and retested. Values that are flagged "HI are inaccurate and should not be reported. 3. Check the instrument printout for flags or error messages before reporting results. 4. For failed curve fit [ <0 990), repeat the assay 5. The range for the control is found on the package insert provided with the Percent Fat Oil Control. Theassay value for the control should approximate this range. The control result should be multiplied by 400 to get the result However, it is advised that each lab stablish a control range using its own equipment and personnel. Limitations New food matrinces with no established protocols are considered special applications and should be checked for intelarference and spike recovery Refer to the Sample Preparation table found in the Saffeast M Binder. Applications For special applications to this assay, call the SAl Product Support Number 1-888-321-SAFE. 32 3-5En-V r 0 b _ 4 1K a, ge > *3 -0 0 CL c cm .09 2 cr 0 0o C11 .0 U 5 t ILt . LC 1- 91 0,: m R Lzi ' 15 & , .3-,, ;, > g R I CL C: I L. a M 0 'a 5 * C, C C, D 33 Percent Fat(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 Fat' 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 S;urnplh Prepwrt?ion Quick S;t rt ar * 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 -3sunJple Pr-epnrjutin CQui.k ,art rc;dfor sample preparation instructions. Pre-Assay Preparation * Label a new set of test tubes: Reagent Blank and C1 through C5 for the calibrators, and 1, 2, etc. for the samples. * Transfer 20 pli of each calibrator and 20 pli 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 0 C. NOTE: If necessary, prepare an additional 1:2 dilution on the initial filtrate. This will double the sample dilution. For example, if the dilution of the initial filtrate was 1:100, the final dilu tion would then be 1:200. . Turn on the SafTestTM' Analyzer and place the 550/690 filter in the filter compartment. * Select the FAT/MSA program and at the 'Blank Tube' prompt, insert a water blank. * After 10 minutes, wipe tubes and insert the reagent blank and then calibrators 1 through 57 as prompted by the SafTesi t M Analyzer. * Wipe and insert the sample tubes in the designated order for analysis and results. 34 APPENDIX C STD Kit Assay Instructions Standard Kit Introduction The Safety Associates, Inc. ProteSafe" STD assay measures the protein concentration in oil samples as milligram perdeciliter(mg/dl) orppm(10ppm is 1 mg/dL) 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 500ml reagent bottle labeled *ProteSafeT Elution Buffer" and set the volume to 500pl. 3. Attach the dispenser labeled "ProteSafe T M A onto the 500 ml reagent bottle labeled "ProteSafeTM Reagent A". The volume is fixed at 1.0ml. so no adjustment is necessary. 4. Attach the dispenser labeled "ProteSafe'M B" onto the 250ml reagent bottle labeled "ProteSafeTM Reagent B." The'volume is fixed at 150pl. so no adjustment is necessary. 5. 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 yellow Q Membrane Units (QMU) with the sample numbers. 2. Turn on the SafTest Tm Analyzer and allow it to warm up. 3. Using the adjustable pipette, transfer 300pl 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 SafTest

T

M 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 (QMU) to the membrane holder. The QMU labels should correspond to the labels on the test tubes. Uncap the QMU's. 35 Assay Performance 1. Prepare a 1:2 dilution of the oil sample by pipetting 1.2ml of oil sample into a labeled test tube. Set the preparation reagent dispenser to 1.2ml 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 iml 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. Turn on the SafTestTM 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 SafTestT M Filtration Unit. Re-attach the MSU and QMUs. Uncap the QMUs. 6. Using the bottle-top dispenser, dispense 500 pl of Elution Buffer to each QMU. Turn on the SarTestm Filtration Unit and filter the samples at 2-5psi. Turn off vacuum. 7. Repeat step 6. Do not change tubes. 8. Remove the MSU with the QMUs. Remove the test tubes and place them in a test tube rack. 9. Aliquot 300ul of each sample from step 8 into a new set of labeled tubes. 10. Dispense one aliquot of ProteSafeTw Reagent A into each calibrator and each sample tube. 11. Dispense one aliquot of ProteSafeTM Reagent B into each calibrator and sample tube. 12. Cap all of the tubes and INVERT GENTLY 3-4 TIMES. Let tubes stand for 2 minutes then read. 13. Select "PROISTD" program in the SafTest T M Analyzer. Place the 5701690 filter in the filter compartment and blank the analyzer by inserting a water blank. 14. Insert Calibrators 1 through 4 as prompted by the SafTest Tm Analyzer. Insert the sample test tubes in the designated order. Wipe each test tube with a lint-free tissue prior to insertion in the SafTestTM Analyzer. Reporting Results 1. The SafTesTmAnalyzer 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 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 (%CV) is expected with samples that are measured at the low end of the calibration curve. 6. For failed curve fit (r' < 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 lab establish control ranges using their own equipment and personnel. 37 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 protein 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 first 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 12ml 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 500pl aliquots using a lml 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 15ml 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 5ml 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 5ml of diluted samples on same membrane and filter. Total volume of filtered sample is 10ml. Discard filtrates and replace tubes with a new set of labeled tubes. Using the elution buffer, wash the membranes two times with 500ul aliquots. Collect the wash filtrates for analysis. 38 Calibration Assay and Subsequent Testing 1. Label a new set of test tubes: Cl through C5 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 turn on the Saffest TM 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 room 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 SajTesrM 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 SafTest" 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 SafTest" M Analyzer. 8. Discard all used reagents. 39 Reporting Results 1. The SafTesf 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: SafTesrM Result Dilution Rsl Dilution x Result Final Result Factor ppm ppm 0.0018% control is prepared at 1:2 9ppm 2x9ppm 18ppm and 10ml supplied Sample prepared the 4ppm 2x4ppm 8ppm 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. Range 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. 40 Protein in Refined Oil Kit (PRO-HSY) Purpose The PercentSeawnih assay is tended in deterrne the arnmount of seasonuig on a chip Principle The Percent Seasomng assay measures tihe protein level m seasoning an chips using a micloprotein assay with colormotc endpoints. The protein concentralion is quantitated in mnilligrams (mg) per dL and converted to % seasoning by comparison to a 6% equivalent seasoning control. Kit Contents The Percent Seasoning lut comes packaged with all the reagents and membrane units needed to run the assay. The it contains Reagent A. Reagent 8. packets to prepare Preparation reagent, one Calibrator Series, and one Control. Each lkt comes with the appropriate number of disposable membrane holders and membrane units. The kit should be stored at 4"C.unti used and left out alter lirst use for up to one month at room temperature. Saifetry Precauton Handle with care. The Poercent Seasuring kit wt stain clothing, hands, and equipment When used as directed, the reagents, controls, and calibrators should present no hazard to the user As a normal laboratory precaution, avoid contact with eyes and skin and wash copiously if on skin or in eyes. On not binaet bw mouth. Do rnot ingest, as ona component is toxic. Materials Required Production Packaqge; Safleaf Workstation: Disposable Labware: Two BokIttle-Top Dispensers Safresf- Analyzer Klmwipes One Reagent Holder Safresr* Filrabon Unit 12 mm Glass Test Tubes Test Tube Caps Adjustable Pipeee Pipe- Tips 13 x 7 5mm Test Tubes 00ril bottles anmi caps Multif Display Tner Plastic baggies Reagent? Dispenser Preparation NOTE: Bnring reagents and dispensers to room temperature (18-25*C) before beginmrning assay. I. Mix the contents of the reagent bottles by gently swirling prior to watching dispensers. 2. Attach the dispenser labeled "Seasoning A* onto the reagent bottle labeled "Percent Seasoning Reagent A' and for "Seasoning 8' onto the reagent bottle labeled "Percent Seasoning Reaagent B. The volumes are preset to the specified volumes in the Pescent Seasomninginstruction section in the Saffet"T Binder. 3 To prepare Preparation Reagent add the contents of one packet marked "Preparation Reagent Sallts" into 5Lters of disiled water. 4. Mis tfor 5 minutes using a stir bar in a large beaker. Save for use in assays throughout the day. 5. ODispense approumrnately I to 2 aliquots of reagents A and B into a waste container to eliminate any air bubbles in the dispensers prior to use. Sample Preparation Sample preparation will vary based on the particular seasoning being tested. Prepare andfor dilute the chip sample appropriately, according to the sample preparation section in the Safestn m Quick Start Card. Pre-Assay Preparation 1. At the first assay each day. label a new set of last tubes: one for each of the calibrators, one tube for the control, and 1. 2, 3, etc. for the samples. If running replicates. label the appropnate amount of tubes for each control and sample. 2. Using the adjustable pipelte. transfer the appropate volume of each calibrator, control, and sample into the designated test tubes. Refer to the Percent Seasoning instruction section in the SafrTest" Binder for aliquoating volumes Wipe the pipette tip pnor to dispensing all samples, controls, and calibrators. Use a new hp for each sample. reagent blank, control., and calibrator. 3. After the first calibraton Assay. use the STAT mode and run one control and one chip sample each time poinL NOTE. Run at least one control with each sample. The control will ensure that the assay is being performed correctly and that the chip sample is within expected % seasoning range. 41 Test Method 1. For the first assay of the day turn on the Saftesfi Analyzer tor 5 minutes Place the T570/S0 filter in the hi ter compartment. Select the "SEAFL program. 2. DEspense one aliquot of Percent Seasoning Reagent A into each of the calibrator tubes. the control tube. and each of the sample tubes. Dispense one arquot of Parent Seasoning Reagent B into each of the calibrator tubes, the control lube. and each of the sample tubes. 3. Cap all the tubes and invemr gently 3.5 times to m. 4. Set timer for 2 m sutea. For the first assay turn on the Saffesf* Analyzer for S minutes. Place the 570/60 flter in the filter compartmenL Select the "SEA/FL" program. S. Zero the instrument by inserting a tube containing water blank. Calibrate the Itatniment by inserting the cahibrators in the proper ceder as prompted by the SafTesrm analyzer. 0. Follow g successful calibralston, insert the control tube. The control should be n every bme the samples are run. To ensure the instrument and reagents are performing properly, the control value should fall within the range stated on the package insert for the lot of controls used (see Package insert -Control for Percent Seasoning Kits). II the control falls significanlly outside the range. rerun the assay. 7. l the control value falls within the range, insert the sample tubes in the designated order to analyze in the SafTestm Analyzer. Wipe each test tube with a lintike tissue prior to insertion in the Saffesr"' Analyrer. 8. At the end of the day, store calibrators, control n the refugerator, and reagent bottles with dispensers attached at room temperature Reporting Results I. The SafTes tm Analyzer wim) use he calibrators to calculate the protein concentration in mgtdi. of controls and the solubilized chip. 2. If the sample value is greater than the value of the highest caklibrator. the instrument wilt flag the result as 'HL' The sample must be diluted at a higher dilution and retested. Values that are flagged 'HI are inaccurate and should not be reported. 3. Check the instrument printout for flags or error messages before reporting results. 4. For landed curve lit (i < 0.99gg0)., repeal the assay. S. The range for the control is found on the package insert provided with the Percent Seasoning Control. The assay value for the central should apprldmate this range. Limitations New food marices with no established protocols are considered special applications and should be checked for interference and spike recovery Refer to the Sample Preparation table found in the Sffest"re" Binder. Applications For special applications to this assay. cal the SAI Product Support Number f .888-321-SAFE. 42

Claims

CLAIMSWhat is claimed is:

1. A system for determining the presence of an analyte in a sample which contains matter other than said analyte, said system comprising: an analyte capture membrane which is operative to capture an analyte; an eluant for eluting the analyte from the capture membrane so as to provide an eluant/analyte admixture; and, at least one reagent which is combinable with the eluant/analyte admixture to provide a reagent/eluant/analyte admixture from which said analyte may be determined.

2. A system according to Claim 1 further comprising: a first membrane which is operative to prevent extraneous matter contained in the sample from passing therethrough, while allowing a filtrate containing said analyte to pass therethrough and subsequently through the analyte capturing membrane.

3. A system according to Claim 1 wherein: the analyte is an inorganic acid; the analyte capturing membrane incorporates negatively charged groups which bind the inorganic acid; the eluant comprises a salt which causes the inorganic acid to be released from the membrane and the at least one reagent comprises a reactive agent which reacts with the inorganic acid in a manner that permits detection of the inorganic acid.

4. A system according to Claim 3 wherein the analyte is citric acid, and wherein: the analyte capturing membrane comprises an anionic membrane ; the eluant comprises 0.5 M NaCI ; and the reagent comprises a solution of sulfanilic acid and sodium nitrite which is combined with the eluant after the eluant passes through the analyte capture membrane.

5. A system according to claim 1 wherein the analyte capture membrane is a first analyte capture membrane, and further comprising: a second analyte capture membrane located upstream of the first analyte capture membrane such that the sample passes through the second analyte capture membrane before passing through the first analyte capture membrane, the second analyte capture membrane being operative to capture an analyte other than the analyte captured by the first analyte capture membrane.

6. A system according to claim 5, wherein the second analyte capture membrane is a weakly basic membrane that retains weak inorganic acids; and the first analyte capture membrane is a strongly basic membrane that retains strong inorganic acids.

7. A system according to claim 6, wherein the second analyte capture membrane is structured to retain acetic acid; and the first analyte capture membrane is structured to retain citric acid.

8. A system according to claim 7 wherein the reagent is a first reagent comprising a solution of sulfanilic acid and sodium nitrite which is combined with the eluant after the eluant passes through the first analyte capture membrane, and further comprising: a second reagent which comprises xylenol orange in neutralized isopropanol which is combined with the eluant after the eluant passes through the second analyte capture membrane.

9. A system according to claim 1 , further comprising an acidity detector operative to detect the acidity of the sample after the sample has passed through the analyte capture membrane.

10. A system according to claim 2 wherein the analyte capture membrane is operative to bind proteins present in the sample, and further comprising a second analyte capture membrane which is operative to bind surfactants present in the sample.

11. A system according to claim 10, wherein the at least one reagent comprises an enzyme that reacts with lipids to separate glycerol from the free fatty acids of the lipids, and comprises at least one other enzyme that reacts with the separated glycerol to produce a detectable signal.

12. A system according to claim 11 wherein the at least one reagent comprises glycerol kinase, adenosine thphosphate, glycerol-1 phosphates, aminoantipyrine, and peroxidase.

13. A system according to claim 1 wherein the analyte capture membrane is operative to bind proteins contained in the sample; the eluant comprises a salt solution; and the at least one reagent comprises an indicator solution that produces a detectable signal when mixed with protein.

14. A system according to claim 13, wherein the at least one reagent comprises a solution containing brilliant blue, methanol, and phosphoric acid.

15. A system according to claim 14 further comprising a spectrometer operative to detect the presence of a detectable signal at a desired wavelength of light.

16. A system according to claim 1 wherein the analyte capture membrane is structured to bind nucleic acids contained in the sample; the eluant comprises a salt solution; and the at least one reagent comprises at least one nucleic acid primer having a nucleotide sequence that is complementary to at least one nucleotide sequence of the nucleic acids retained by the analyte capture membrane.

17. A system according to claim 16, further comprising at least one additional analyte capture membrane structured to bind nucleic acids contained in the sample, each of the membranes being serially arranged such that the sample passes through one of the analyte capture membranes before passing through another analyte capture membrane.

18. A system according to claim 16 wherein the analyte capture membrane is an anionic membrane.

19. A system according to claim 16 wherein the analyte capture membrane comprises antibodies structured to bind to nucleic acids, and being selective for nucleic acids of a desired microbe.

20. A system according to claim 16 further comprising a device to perform a polymerase chain reaction using the nucleic acid primers of the at least one reagent and the nucleic acids obtained from the sample.

21. A system according to claim 16, further comprising a first membrane which is operative to prevent extraneous matter contained in the sample from passing therethrough, while allowing a filtrate containing said analyte to pass therethrough and subsequently through the analyte capturing membrane.

22. A system for determining the presence of an analyte in a matrix which contains matter other than said analyte and an interferant which interferes with the analytical test to be used to determine the presence of the analyte, said system comprising: at least one interferant-capturing membrane which is operative to capture the interferant while allowing a filtrate which contains the analyte but is substantially devoid of the interferant to pass therethrough; and, a reagent which is combineable with the filtrate containg the analyte to provide a filtrate/reagent/analyte admixture from which the analyte may be determined.

23. A system according to claim 22 further comprising a first membrane which is operative to prevent some of the matter of said matrix from passing therethrough, while allowing a filtrate containing the interferant and the analyte to pass therethrough.

24. A system according to 22 for additionally analyzing the interferant, said system further comprising: an eluant for eluting the interferant from the interferant-capturing membrane so as to provide an eluant/interferant admixture; and a second reagent that is combinable with the eluant/interferant admixture to provide an eluant/interferant/reagent admixture from which the interferent can be determined.

25. A system according to 22 wherein the analyte is free fatty acid and the interferant is an inorganic acid, and wherein: the interferant-capturing membrane comprises an anionic membrane which captures inorganic acids; and, the reagent for determining the presence of the analyte comprises xylenol orange and neutralized isopropanol.

26. A system according to 25 further comprising: an eluant which is useable to elute the inorganic acid from the interferant- capturing membrane to provide an eluant/inorganic acid admixture; and, a second reagent that is combinable with the eluant reagent admixture to facilitate determination of the inorganic acid.

27. A system according to 22 wherein the at least one interferant-capturing membrane comprises: a first interferant-capturing membrane that is operative to capture a portion of the interferant; and, a second interferant-capturing membrane that is operative to capture a second portion of the interferant.

28. A system according to claim 27 wherein the first interferant-capturing membrane is operative to capture a weak inorganic acid, and wherein the second interferant-capturing membrane is operative to capture a strong inorganic acid.

29. A system according to claim 28 wherein the first interferant-capturing membrane is operative to capture acetic acid, and wherein the second interferant- capturing membrane is opertative to capture phosphoric acid, and wherein the reagent reacts with free fatty acids filtered from the matrix to produce a detectable signal.

30. A system for detecting the presence of a microbe in a sample, comprising at least one analyte capture membrane operative to capture nucleic acids contained in the sample; an eluant for eluting the nucleic acids from the capture membrane so as to provide an eluant/nucleic acid admixture; and at least one nuclec acid primer structured to hybridize to nucleic acids of microbes suspected of being present in the sample and provided in an amount to facilitate amplification of the nucleic acids that hybridize to the nucleic acid primers so that the nucleic acids of the microbes can be detected.

31. A system according to claim 30, comprising a plurality of analyte capturing membranes.

1 32. A system according to claim 30, wherein the analyte capturing membrane

2 comprises an anionic membrane.

1 33. A system according to claim 30, wherein the analyte capturing membrane

2 comprises antibodies attached to the membrane and being operative to bind to the

3 nucleic acids in the sample.

1 34. A system according to claim 33, wherein the analyte capturing membrane

2 comprises antibodies attached to the membrane and being operative to bind to DNA

3 of a microbe suspected of being present in the sample.

l

35. A system according to claim 30 wherein the eluant comprises a salt solution.

l

36. A system according to claim 35 wherein the eluant comprises 1 M NaCI.

1 37. A system according to claim 30, further comprising a particulate removal

2 membrane positioned with respect to the analyte capture membrane such that the

3 sample passes through the particulate removal membrane to retain particles of the

4 sample, and to pass the nucleic acids in the sample to the analyte capture

5 membrane.

1 38. A system according to claim 30, further comprising a homogenizer that is

2 operative to homogenize the sample before the sample is applied to the membrane.

39. A system according to claim 30, further comprising a device that is operative to amplify the nucleic acids using a polymerase chain reaction.

40. A system according to claim 30, further comprising a sensor to detect a signal indicating the presence of a nucleic acid of a microbe in the sample.

41. A system according to claim 30, wherein the nucleic acid primers are structured to hybridize to a nucleic acid of e coli bacteria.

42. A system according to claim 41 , wherein the nucleic acid primers are structured to hybridize to a nucleic acid of e coli H157.