EP1463744A4

EP1463744A4 - Devices and methods for isolating and detecting specific substances in complex matrices

Info

Publication number: EP1463744A4
Application number: EP02805656A
Authority: EP
Inventors: Virginia C Gordon
Original assignee: SAFETEST Inc
Current assignee: SAFETEST Inc
Priority date: 2001-12-20
Filing date: 2002-12-20
Publication date: 2005-10-26
Also published as: CA2470724A1; EP1463744A2; WO2003054516A2; WO2003054516A3; AU2002365053A1; JP2005533239A

Abstract

Methods and systems for determining the presence of one or more analytes in various types of matricies,such as foods, oils, biological fluids, etc. Membranes are used to remove extraneous matter (e.g., solid particles) and/or to bind specific interferents or analytes. Specific assays are provided for determining citric acid, total fat (or % fat), free fatty acids, protein content and specific nucleic acid content indicative of the presence of DNA and/or RNA of certain microbes (e.g., pathogens) or genetically altered substances (e.g., genetically engineered grain).

Description

DEVICES AND METHODS FOR ISOLATING AND DETECTING SPECIFIC SUBSTANCES IN COMPLEX MATRICES

Related Applications 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 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.

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.

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 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 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 and/or chemical species, some or all of which may interfere with the intended analysis. Thus, it is frequently necessary for the test substance to be subjected to extensive sample preparation steps, in order to isolate and/or concentrate the particular analyte(s) of interest, prior to actually proceeding with analytical determination of the desired analyte(s). Moreover, in instances where the test substance is a solid material (e.g., food) it is often necessary to chop or grind the solid material into particles, and to extract the desired analyte(s) from such particles by adding one or more liquid digestants, solvents or other fluids to form a slurry or suspension, and thereafter performing a "clean up" of the slurry or suspension by filtration or centrifugation to separate the analyte containing liquid from the extraneous solid matter.

In instances where multiple analytes are to be determined, it is often necessary to perform several separate, time consuming, analytical procedures (e.g., gas chromatography (GC), high performance liquid chromatography (HPLC) or other analytical chemistry procedures) on aliquots or extracts of the test substance, in order to generate the desired multiple analyte data.

Thus, the traditional methods for determining the presence of, or detecting specific analyte(s) in complex matrices (e.g., substances which contain matter other than the desired analyze(s)) can be quite time consuming, skill intensive and expensive.

It is frequently desirable to detect or quantify, in foods, one or more particular analyte(s) which are indicative of the freshness or quality of the food. In routine quality control testing of foods, it is common practice to test for the presence of various contaminates, additives, degradation products, and/or chemical markers of microbial infestation (e.g., bacterial endotoxins, mycotoxins, etc.). However, the current methods by which such quality control testing of food is accomplished are typically either: a) complex and skill-intensive analytical chemistry procedures or b) highly subjective and qualitative sensory evaluations (e.g., smell test, taste test, appearance, etc.). 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 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 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 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 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. 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 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. 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. Summary of the Invention

The present invention provides methods and systems (e.g., test kits) for qualitative and/or quantitative determination of one or more analytes present within a matrix that contains matter other than the analyte (e.g., solids, particulate matter, matter or substances that will interfere with the analysis, etc.). These methods and apparatus are useable to detect or quantify specific analytes present in complex matrices such as foods, cosmetics or biologicals, organ/tissue homogenates, industrial waste, sewage, industrial fluids, microbiological or pharmaceutical incubator slurries, etc. to determine the quality, degradation, age, abuse, contamination, nutritional value, purity and other characteristics of the matrices.

In accordance with this invention, there is provided a method and system (e.g., test kit) for determining the presence of a single analyte. This system comprises; a) a sample receiving vessel, b) a membrane and c) a reagent-containing well. The test sample is initially prepared (e.g., chopped or ground if a solid) and is deposited in the sample-receiving vessel along with any desired diluent, digestion solution (e.g., enzymes), chelators, or chemical modifiers (e.g., antioxidants). The prepared sample is then permitted to drain from the sample-receiving vessel, through the membrane. The type of membrane utilized in each embodiment will be selected based on the type and quantity of matter which is desired to be excluded from the prepared sample matter prior to analysis. In many applications, this initial membrane will be formed of microporous film having pores which are sized to present large particles of solid matter, proteins and other unwanted matter from passing therethrough, but which will allow a filtrate containing the desired analyte to drain into the reagent-containing well. When drained into reagent-containing well, the analyte contained within the filtrate will react with the reagent in a manner which will permit the presence or quantity of analyte to be determined. In many instances, the analyte-reagent reaction will be a color forming reaction such that a visual determination may be made as to whether, or to what degree the desired analyte is present. In other instances, it may be desirable to utilize an analytical instrument to 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 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. 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 therethrough. Each of these additional membranes may subsequently be exposed to a wash or flush solution such that one or more eluants containing each of the additional analytes may be obtained. Each such eluant may subsequently be combined with a reagent to provide an eluant-reagent admixture from which at least one analyte may be determined. In this manner, the present invention is adaptable for the qualitative or quantitative determination of two or more analytes from a single sample.

Further in accordance with this invention, in situations where one or more analytes is/are present in a matrix at low concentrations (e.g., concentrations that are below the detection limit of the intended analytical test) the analyte may be captured on a membrane and may be subsequently eluted from that membrane with a volume of eluent that is substantially smaller than the volume of the original sample, thereby providing an analyte/eluant admixture wherein the concentration of the analyte is sufficiently high to permit its detection by the intended analytical method. The starting concentration of the analyte in the original sample may then be determined by calculation based on the known volume of the original sample and the known volume of the eluant that was used to elute the analyte from the membrane.

Further in accordance with this invention, there are provided methods and systems of the foregoing character wherein a membrane is used to remove a positive or negative interferant from the sample to permit an analyte to be analyzed 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 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 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 inorganic acid that was present in the sample. In such situations, an eluant that releases the inorganic acid from the negatively charged membrane is used to elute the inorganic acid from the membrane on which it was captured, thereby providing an inorganic acid/eluant admixture from which the inorganic acid may be quantitatively or qualitatively analyzed. In some situations it may be additionally desirable to desperate specific types of inorganic acids present in the sample and to analyze for one or both of those types of inorganic acids. Accordingly, in such instances, the sample may be passed through a plurality of membranes, each of which has a binding affinity for a different type of inorganic acid, before the filtrate is analyzed for FFA. In this regard, a first membrane may be impregnated or coated 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 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 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 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 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 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 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 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 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 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. Figure 5 is a table listing specific test methods and systems and specifying the analytes, typical matricies in which the analyte is contained, specific membranes (cross-referenced to Figure 3) and the specific detection reagents (cross-referenced to Figure 4) useable in each test method and system. Detailed Description

The following detailed description and the figures to which it refers are not intended to describe all possible embodiments and examples of the invention. Rather, this detailed description and the accompanying figures are directed to certain illustrative embodiments and examples of the invention only and does not limit the 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 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 the Saftest™ Membrane Unit and the Saftest™ 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 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 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 extraneous matter is retained above the membrane and a filtrate 16 containing the analyte passes through the filtration membrane 13 and into the filtrate collection well 16. A desired analytical or detection technique may then be used to quantitatively or qualitatively determine the analyte in the filtrate 20. In some instances, such analysis will require one or more reagents to be mixed with the analyte-containing filtrate 20. In other instances, the neat filtrate 20 may be used for the analysis (e.g., examined microscopically, placed in an analytical instrument such as a spectrophotometer or chromatograph or applied to an indicator (e.g., pH paper, paper or dip sticks which indicate the presence of the analyte, etc.) In other embodiments, the sample18 may be substantially free of extraneous matter that must be removed by a filtration membrane 13 (e.g., a clean oil or liquid solution) but, instead, the sample 18 may contain two analytes that must be separated or some interferant that will interfere with analysis for the analyte and must therefore be separated from the analyte prior to analysis. In these embodiments, a capture membrane 14 will be mounted on the membrane support rather than a filtration membrane 13. This capture membrane 14 may be selected so as to capture (e.g., chemically bond to or otherwise hold) a first analyte while allowing a second analyte to pass therethrough in the filtrate 20. The first analyte may subsequently be eluted (e.g., released) from the capture membrane and determined separately and the first analyte contained in the filtrate 20 may also be determined. The capture membrane may also be used to capture an interferant while allowing a filtrate containg the analyte to pass therethrough or vice versa.

Figure 2 shows, in schematic fashion, a two membrane device . Here, the top membrane is either a filtration membrane 13 (for samples 18 which contain extraneous matter that must be filtered out) or a capture membrane 14 (for samples that contain multiple analytes or interferants). The bottom membrane is a capture membrane 14. The sample 18 passes through the top membrane which removes extraneous matter or captures a first analyte or interferant. The filtrate that has passed through the top membrane then passes through the bottom membrane which 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 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 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 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 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 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 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 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 the lipids are dissolved in a liquid phase. A first membrane may be used to remove solid extraneous matter. The liquid, lipid-containing filtrate is then passed through the capture membrane such that the citric acid becomes bound to the capture membrane. The free fatty acids are then measured in the filtrate that passes through the capture membrane. The citric acid is then released from the capture membrane by elution with a salt solution as described above. The eluant/citric acid admixture may then be contained in a second vessel and the presence and/or amount of citric acid may be analyzed as described above.

2. A method and system for determining acetic acid and free fatty acid can be used for determinations in other food matrices and encapsulated lipids in foods where the food is solubilized. In the same manner as the citric acid assay described above, a first filtration membrane is used to remove particles and other solid matter. The acetic acid containg bound to the capture membrane, the free fatty acids 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 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 compounds formed in the degradation of shrimp..

6. Any of the above kits s to be used in conjunction with a second test for malonaldehyde utilizing a methyl indole reagent with weak acid such as small amount of HCI.

7. Any of the above kits to be used with a second test for lipid peroxides in the eluant using an iron catalyzed electron transfer to xylenol orange. 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.

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 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 1ml.

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 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.

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 thglycehde content.

14. A test method and kit to determine oxidation of beverages and determination efficacy of certain additives and/or stabilizers on oxidation using the alkenal test The beverage, carbonated or not, is separated through protein binding membrane and the filtrate tested with methylindolis solution with sulfonic acid.

15. A test for rapid determination of the quality of cooking oils and fats by testing for lipid peroxides using a peroxidase and iron catalyzed reagent and complexed to 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.

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 performed.

17. A test method and kit to detect afiatoxins in foods or tissues by emulsifying the food and releasing the afiatoxins using surfactants or osmotic changes to lyse the membranes and cells and after filtering out particulates using a second membrane coated with an antibody specific to multiple or particular afiatoxins. The afiatoxins 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 osmotic changes to lyse the membranes and cells and using a particulate binding membrane followed by a ribonucleic acid binding membrane. The RNA is released and then amplification of a sequence specific to the target organism to detect its presence performed.

Detailed Examples Of Specific Embodiments Of The Present Invention

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 in commonly owned PCT International Patent Publication No. WO 99/20396 and U.S. Patent No. 6,489,132, and the publicly available SafTest™ 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 reference.

Example 1

Separation and Determination of Free Fatty Acid (FFA) and Citric Acid in an Oil Sample

This example demonstrates free fatty acids contained in an oil sample. The oil sample also contains citric acid. It is desirable to separate the citric acid from the sample prior to assay of the FFA content as the presence of inorganic acids such as citric A 1 ml_ sample of soybean oil is applied to a membrane of a filtering device. The membrane is a strongly basic anionic membrane, such as the Q membrane adsorber membrane with quaternary ammonium groups (Q-MA membrane) publicly available from Sartohus (Sartohus North America, Inc., Edgewood, NY). As the sample is 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 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 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 CitriSafe™ from Saftest, Inc. (Phoenix, AZ). The CitriSafe™ test kit is generally described in Appendix A to this patent application.

The amount of free fatty acids originally present in the soybean oil is determined by measuring the acidity of the oil after the removal of the citric acid using the methodology described in incorporated parent application Serial No. 09/183,157 and commercially available as a test kit under the name FASafe™ from Saftest, Inc. (Phoenix, AZ).

The CitriSafe™ and FASafe™ test kits are 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 devices are commercially available as the Saftest™ Membrane

Unit and the Saftest™ Filtration Unit or Saftest™ Work Station, from Saftest, Inc.

Example 2

Removal of Interfering Inorganic Acids and Determination of Free Fatty Acid (FFA) and Acetic Acid in a Food Sample This example demonstrates the separation of inorganic acids from free fatty acids in a food sample and the subsequent determination of acetic acid and free fatty acid content of that food sample.

A 5 gram sample of mackerel is solubilized to create a slurry. The slurry is heated to approximately 40-45 °C and filtered to remove particulates from the slurry. Two (2) mL of the filtered slurry is applied to a membrane structure of a filtering device. The membrane structure includes two stacked membranes one disposed on top of the other. The upper membrane is a weakly basic membrane, such as the D 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 Sartohus (Sartorius North America, Inc., Edgewood, NY). As the filtered slurry is applied to the membrane structure, acetic 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. 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 μl 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 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. 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 FASafe™ publicly available from Saftest, Inc. (Phoenix, AZ). The FASafe™ 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 devices are commercially available as the Saftest™ Membrane Unit and the Saftest™ Filtration Unit or Saftest™ Work Station, from Saftest, Inc.

Example 3

Determination of Total Fat Content and/or Percent Fat

This example demonstrates the determination of fat content or 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 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 Sartohus, Inc, and then the filtered homogenate is passed through a membrane that binds surfactants, such as the MA-Q or MA-S membranes from Sartorius. The MA-S membrane has sulfonyl groups on the membrane surface for binding surfactants.

A portion of the filtrate (20 μL) 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. 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 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 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 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 Saftest™ Membrane Unit and the Saftest™ Filtration Unit or Saftest™ Work Station, from Saftest, Inc.

Example 4

Determination of Protein Content in Refined Oil 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 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 buffered, low salt solution (0.05 M NaCI in phosphate buffer at a pH between 7 and 9). One (1) mL of the concentrated filtrate is mixed with

0.3 mL of an indicator solution containing 0.1% brilliant blue (Sigma, St.

Louis, MO) in 30% methanol, and 0.3% phosphoric acid for two minutes at room temperature (18-25 °C). The presence of protein is 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 as ProteSafe™ from Saftest, Inc. (Phoenix, AZ) and is described in

Appendix C to this patent application. The ProteSafe™ 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 devices are commercially available as the Saftest™ Membrane Unit and the Saftest™ Filtration Unit or Saftest™

Work Station, from Saftest, Inc.

Example 5

Determining the Presence Of A Particular Microbe in a Food Sample 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 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 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 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 (Sartorius), which are reacted with the protein amino groups of the antibodies. The slurry is passed through two additional nucleic acid binding membranes to increase the amount of nucleic acid removed from the homogenate. The filtrate is then discarded.

The membranes are washed with 1 M NaCI in water to release the nucleic acids from the membranes into a container. The RNA and DNA are then amplified using polymerase chain reaction (PCR) and one or more nucleic acid primers that have sequences for ecoli H157. PCR methods are conventionally known to persons of ordinary skill in the art, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3^rd Edition, 2001. The PCR products are labeled by incorporating a fluorescent marker during the amplification steps, and the presence of ecoli H157 is determined by measuring the fluorescence contained in the PCR products.

Although exemplary embodiments of the invention have been shown and described above, many changes, modifications, substitutions, variations and/or additions may be made by those having ordinary skill in the art without necessarily departing from the spirit and scope of this invention. For example, where this patent application has described the performance of steps of a method or procedure in a specific order, it may be possible (or even expedient in certain circumstances) to 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 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. APPENDIX A

CitriSafe -"STD Kit Assay Instructions Standard Kit

Introduction

The Safety Associates, Inc. CitriSafe™ 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 Buffe and set the volume to 750 μl.

3. Attach the dispenser labeled in yellow as "CitriSafe A" onto the 500 ml reagent bottle labeled "CitriSafe Reagent A" and set the volume to 1.5 ml.

4. Attach the dispenser labeled in yellow as "CitriSafe B" onto the 250 ml reagent bottle labeled "CitriSafe Reagent B.° The volume is fixed at 100 μl, 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 μl of the reagent blank and 750 μl 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. Set up the SafTest™ 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- embrane 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 sample. 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 SafTest™ 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 SafTesf™ Filtration Unit. Reattach the MSU and QMUs. Uncap the QMUs.

6. Using the bottle-top dispenser, dispense 750 μl of Elution Buffer to each QMU. Turn on the SafTest™ 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 (100μl) 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 SafTesf™ Analyzer and allow it to warm up. Place the 420/690 filter in the filter compartment. Select "CIT/STD" program.

13. After 30 the minute heating period, blank the SafTest™ 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™ Analyzer, it is unacceptable and must be repeated.

14. Insert Standards 1 through 3 as prompted by the SafTest™ Analyzer. Insert the sample test tubes in the designated order. Wipe each test tube with a lint-free tissue prior to insertion in the SafTest™ Analyzer. Reporting Results

. The SarTesf¹* 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.

Kit for Oils

Purpose

Principle

Reagent/ Dispenser Preparation

Pre-Assay Preparation

Reporting Results

Limitations

Applications

APPENDIX B

Percent Fat Kit MSA for pet food, rendered meals, seeds, nuts and nut butter

Purpose

The Percent Fat assay is intended to determine the amount ol lipid or (at in a sample.

Principle

The Percent Fat assay measures the (at level in samples using eπzymalic hydrolysis ol triglycendes to glycerol. followed by an eπzymaticcolonmetric measurement ol the glycerol released. The tπglyceπde concentration is quaπlitated in grams (g) per 100 grams ol sample.

Kit Contents The Percent Fat kit comes packaged with all the reagents and membrane units needed to run the assay. The kit contains Reagent A. one Calibrator Seπes. and one Control. Each kit comes with the appropriate number ol disposable membrane holders and membrane units. The kit should be stored at 4*C.

Safety Precaution: Handle with care. The Percent Fat reagent will stain clothing and equipment. When used as directed, the Percent Far reagents, controls, and calibrators should present no hazard to the user. As a normal laboratory precaution, avoid contact with eyes and skin. Do not oioel by mouth.

Materials Required

Production Pacfcaoe: SafTeef • Workstation: Disposable Labware: One Bottle-Top Dispensers SafTesf Analyzer Kim wipes One Reagent Holder SafTesf Filtration Unit 12 mm Glass Test Tubes

Heat Block Test Tube Caps

Vortex Pipette Tips

Positive Displacement Pipette

Adjustable Pipette

13 x75mm Test Tube

Multi Display Timer

SAI Heat Source (lor Hydrogeπated Oil

Samples)

Reagent/ Dispenser Preparation

NOTE. Bπng reagents and dispensers to room temperature (1β-25°C) belore beginning assay. 1 Mix the contents ol the reagent bottles by gently swirling prior to attaching dispensers.

2. Attach the dispenser labeled m red as "Percent Fat A* onto the reagent bottle labeled 'Percent Fat A". Dispenser volume is fixed, so no adjustment 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 SatTett"" Binder for Preparation Reagent volume.

4. Dispense approximately 4 to 5 aliquots ol each reagent into a waste container to eliminate any air bubbles in the dispensers poor to use.

Sample Preparation

Sample preparation will van/ based on the particular atnx material being tested. Prepare and/or dilute the sample appropriately, according to the sample preparation section in the SatTett"' Quick Stan 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. II running replicates, label the appropnate amount of tubes lor each control and sample

2. Using the adjustable pipette, transfer Ihβ appropnate volume of each calibrator, reagent blank, control, and sample into the designated tesl tubes. Refer to the Percent Fat assay instruction section in the SafTesf "* Binder for afequoting volumes. Wipe Ihβ pipette lip pnor to dispensing all samples, reagent blank, controls, and calibrators. Use a new tip lor each sample, reagent blank, control, and calibrator.

NOTE: Run at least on* control with each assay. The control win ensure thai the assay is being performed correctly.

Test Method t. 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 aH tubes hi Ihβ heat -Hock set at 37-40"C for the time penod specified m the Percent Fat assay instruction section found in the SafTeat^m Binder

4. Approximately S minutes before the end of the healing penod turn on the SafTesf Analyzer and allow it to warm up. Place the 550/690 filter in the filter compartment. Select the TAT MSA* program.

5. Zero the instnnnent by inserting a tube containing a water blank followed by the Reagent Blank, tf 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 SafTest'" analyzer. β. Following successful calibration, insert the control tube. The control should be run even/ lima the calibrators are run. To ensure the Instrument and reagents are performing property, the control value should fall within the range slated on the package insert for the lot ol controls used (see Package Insert • Control for Percent Fat Kits). It the control fans significantly outside the range, rerun the assay.

7. II the control value falls within the range, insert the sample tubes in the designated order to analyze in the SafTest"' Analyzer. Wipe each test tube with a knt-free tissue pπor to insertion in the SafTest'" 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 SafTest"' Analyzer wiD use the calibrators to calculate the tngtyceridβ concentration in grams (g) of tπgryceπdβ per 100 grams of oil.

2. If the sample value is greater than Ihβ value of the highest calibrator, the instrument will Hag the result as HI.' The sample must be diluted al a higher dilution and retested. Values that are Nagged '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 (r* < 0.990). repeal the assay.

5. The range for the control is found on the package insert provided with the Percent Fat Oil Control The assay value for the control should approximate this range. The control result should be multiplied by 400 to gel the result. However, it is advised that each lab establish a control range using its own equipment and personnel

Limitations

New food matrices with no established protocols are considered special applications and should be checked for interference and spike recovery. Refer to Ihβ Sample Preparation table found In the SafTest"" Binder.

Applications For special applications to this assay, call the SAI Product Support Number I -688-321 -SAFE.

Percent Fat Test Metii '^(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 triglycendes 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 10Oul of the membrane filtrate is combined with 2.4ml 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 μl of each calibrator, 20 μl of each control, and 20 μl 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. Test Method

1. Turn on the SafTest™ Analyzer and allow it to warm up. Place the 550/690 filter in the filter compartment and select the FAT/MSA program.

2. Dispense out 1-2 aliquots of Reagent A, into a waste container to ensure that there are no air bubbles in the nozzles.

3. Dispense one aliquot Percent Fat Reagent A into each of the calibrator, control, and sample tubes.

4. Cap the tubes and gently invert 5 times. Place the tubes in a heat block for 10 minutes (-37-42°C).

NOTE: After 5 minutes, perform a visual check to determine if samples will fall within the range of the curve. To perform a visual check, compare the color of the samples with the color of Calibrator 5. If the sample color is lighter than the color of Calibrator 5, place the sample tubes back on the heat block for the remaining time and proceed to Step 6. However, if the sample color is darker than the color of Calibrator 5, samples must then be further diluted. First, complete the assay for those samples that are lighter than the last calibrator. Then, for those samples that are darker than the last calibrator, begin at Step 5 for instructions for further dilution and testing of out of range samples.

5. If sample colors appeared darker than Calibrator 5 or if the SafTest™ 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 1ml 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 μl 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 SafTest™ 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™ Analyzer.

7. Following successful calibration, insert the control making sure the values of the controls fall within the ranges for the lot of controls used once multiplied by 400 for the dilution (see Package Insert-Controls for Percent Fat Kits). If the controls fall significantly outside the ranges, rerun the assay.

8. If the control values fall within the ranges, insert the sample tubes and analyze in the SafTest™ Analyzer in the designated order (see SafTest™ Analyzer Instructions for detailed instructions). Wipe each test tube with a lint-free tissue prior to insertion in the SafTest™ 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 SafTest™ Analyzer when not in use. Reporting Results

1. The SafTest™ 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:

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 SafTest™ Analyzer Instruction section for detailed instructions on sample analysis using the STAT mode.

Kit STD (mayonnaise)

Purpose

The Percent Fat assay is intended to determine the amount of lipid or rat in a sample

Principle

The Percent Fat assay measures Ihe fat level in samples using enzymatic hydrolysis of triglycendes to glycerol, followed by an enzymaUc colonmetπc measurement of the glycerol released The tnglyceride concentration is quanlitated m grams (g) per 100 grams of sample.

Kit Contents The Percent Fat kit comes packaged with all Ihe reagents and membrane units needed to run the assay. The kit contains Reagent A. one Calibrator Seπes, and one Control Each kit comes with the appropnate number of disposable membrane holders and membrane units The kit should be stored at 4*C

Safety Precaution: Handle with care The Percent Fat reagent win stain ctothing 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 Oo not oioet bv mouth

Materials Required

Production Package; SafTesf" Workstation: Disposable Labware: One Bottle-Top Dispensers SafTesf" Analyzer Kimw es One Reagent Holder SafTesf Filtration Unit 12 mm Glass Test Tubes Heat Block Test Tube Caps Vortei Pipette Tips

Positive Displacement Pipette Adjustable Pipette 13 75mm Test Tube Multi Display Timer

SAI Heat Source (for Hydrogenated Oil Samples)

Reagent Oispenser Preparation

NOTE: Bring reagents and dispensers to room temperature (18-25°C) before beginning assay.

1. Mis the contents of the reagent bottles by gently s irhng pnor to attaching dispensers.

2 Attach Ihe dispenser labeled In red as 'Percent Fat A* onto the reagent bottle labeled 'Percent Fat A" and set Ihe volume to the specified volume in Ihe Percent Fat assay instruction section in the SafTest^,u Binder.

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. Rarer to the SafTest™ 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 and/or dilute Ihe sample appropriately, according to the sample preparation section in the SafTest™ Quick Start Card

Pre-Assay Preparation

1. Label a new set of lest tubes, one for each of Ihe calibrators, RB for the Reagent Blank, one tube for the control, and 1. 2, 3, etc. for the samples If running replicates, label Ihe appropriate amount of tubes for each control and sample.

2. Using the adjustable pipette, transfer the appropnate volume of each calibrator, reagent blank, control, and sample mto the designated test tubes Refer to the Percent Fat assay instruction section in the S-f esf™ Binder for aliquotiπg volumes Wipe the pipette tip prior to dispensing all samples, reagent blank, controls, and calibrators 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.

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 Ihe heat block set at 37-40'C for the lime penod speαfied in the Percent Fat assay instruction section found in the SafTest" Binder

4. Approximately 5 minutes before the end of Ihe mixing penod. turn on the SafTesf"* Analyzer and allow it to warm up Place Ihe 550 690 filter in the filter compartment Select the "FAT/STD" program.

5. Zero Ihe instrument by inserting a tube containing a water blank followed y the Reagent Blank. If the Reagent Blank has an optical density (OD) reader greater than 0200, it is unacceptable and must be repeated Calibrate the instrument by inserting the calibrators in the proper order as prompted by the SafTesf" analyzer.

6. Following successful calibration, insert Ihe control tube. The control should be run every time the calibrators are run. To ensure the instrument and reagents are performing properly, the control value should fall within Ihe range stated on Ihe package insert for the lot of controls used (see Package Insert - Control for Percent Fat Kits). If the control falls significantly outside the range, rerun the assay

7. If the control value falls within the range, insert the sample tubes in the designated order to analyze in the SafTest'" Analyzer. Wipe each test tube with a Imt-free tissue prior to insertion in the SafTesf"* Analyzer. β. 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 SafTest" Analyzer will use the calibrators to calculate Ihe tnglyceride concentration in grams (g) of triglyceπde per 100 grams of oil.

2 if the sample value is greater than the value of the highest calibrator, the instrument will flag Ihe 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.

4. For failed curve fit (r² < 0990), repeat the assay

5. The range for the control is found on the package insert provided with Ihe Percent Fat Oil Control. The assay 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 establish a control range using its own equipment and personnel.

Limitations

New food matrices with no established protocols are considered special applications and should be checked for interference and spike recovery Refer to Ihe Sample Preparation table found in Ihe S_fTe-l^m Binder.

Applications

For βpecial applications to this assay, call the SAI Product Support Number 1-888-321 -SAFE.

Percent Fat (FAT/MSA) Kit Quick Start Card

Matrix Kit

Step 2

Step l ■ Label a (jlass test tube lor each sample and place the tubes inside Ihe acrylic base on the SβtTest™ Filtration Unit

• Consult the Sample Preparation Quick Start Card and add the sample and Preparation Reβgenl In the • Place the membrane holder on the acrylic base. proper proportion to a 15 ml conical tube or glass test • Pour each sample Into the appropriate membrane unit atop the tube. SafT#»r» Filtration Unit

• II necessary, place samples in the prβ-heatβd heat ■ Turn the vacuum on at a setting of S-10 inch Hg block as shown below. • Filter the entire sample through the membrane. Seed samples require multiple washing, therefore, follow seed Instructions. Upon completion, discard the used membrane holder and place the tubes containing the filtrates in a test tube rack.

Step >l

• Place the 5507690 lilter In the λ slot.

• Select the TAT MSA" program and insert the tubes into the SafTeat™ Analyzer in the designated order lor analysis and results.

Percent Fat (FAT/MSA) 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 Faf 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 .irrφln Pn/jstrs n Guic-li J - siri Cu

• 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 S..mpte P/zpurai n Quick J - '.-iri Cvrdfor 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 μl of each calibrator and 20 μl of the samples into the designated tubes.

Test Procedure

• Dispense one aliquot of Percent Fat Reagent A into each calibrator and sample tube.

• Cap tubes and invert 4 times. DO NOT VORTEX. Place tubes in a heat block for 10 minutes at 40°C.

NOTE: If necessary, prepare an additional 1 :2 dilution on the initial filtrate. This will double the sample dilution. For example, if the dilution of the initial filtrate was 1 :100, the final dilu_: tion would then be 1 :200.

• Turn on the SafTesf* 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 -5^* as prompted by the SarTesf™ Analyzer. Wipe and insert the sample tubes in the designated order for analysis and results.

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 per deciliter (mg/dl) or ppm (10ppm is 1 mg/dL)

Dispenser and Reagent Preparation

2. Attach the dispenser labeled "ProteSafe™ Elution Buffer" onto the 500ml reagent bottle labeled "ProteSafe™ Elution Buffer" and set the volume to 500μl.

3. Attach the dispenser labeled "ProteSafe™ A" onto the 500 ml reagent bottle labeled "ProteSafe™ Reagent A". The volume is fixed at 1.0ml, so no adjustment is necessary.

4. Attach the dispenser labeled "ProteSafe™ B" onto the 250ml reagent bottle labeled "ProteSafe™ Reagent B." The'volume is fixed at 150μl. 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™ Analyzer and allow it to warm up.

3. Using the adjustable pipette, transfer 300μl 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™ 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. 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 1ml of the diluted oil sample to each QMU. Use a new tip for each sample. Always wipe the pipette tip prior to dispensing the samples. Recap the QMUs..

3. Turn on the Sa Test™ Filtration Unit and filter the samples. Important. Make sure the vacuum gauge does not exceed 5ρsi 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 SafTest™ Filtration Unit. Re-attach the MSU and QMUs. Uncap the QMUs.

6. Using the bottle-top dispenser, dispense 500μl of Elution Buffer to each QMU. Turn on the SafTest™ 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 ProteSafe™ Reagent A into each calibrator and each sample tube.

11. Dispense one aliquot of ProteSafe™ 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 "PRO/STD" program in the SafTest™ Analyzer. Place the 570/690 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™ Analyzer. Insert the sample test tubes in the designated order. Wipe each test tube with a lint-free tissue prior to insertion in the SafTesf™ Analyzer.

Reporting Results

1. The SafTest™ Analyzer will report results as milligram per deciliter (mg/dl) of protein in the sample. This can be converted to ppm by multiplying by 10.

2. If preliminary dilutions were made to the oil sample prior to testing, the dilution factor must be taken into account. Multiply the instrument results by the dilution factor to obtain the value of the sample. 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)."

6. For failed curve fit (r* < 0.995). repeat the assay.

Protein Content in Refined Oils Assay Instructions -

Introduction

The Safety Associates, Inc. ProteSafe M 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.

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 SOOμl aliquots using a 1ml 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. 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 μl 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 SafTest™ 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 SafTes ^rM 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 SafTesf™¹ Analyzer.

8. Discard all used reagents. Reporting Results

1. The SafTesf™ 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:

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.

Protein in Refined OilYλi (PRO-HSY)

Purpose

The Percent Seasoning assay is intended to determine the amount of seasoning on a chip.

Principle

The Percent Seasoning assay measures the protein level n seasoning on chips using a microprotβin assay with cobjrmetnc endpoints. The protem concentration is quaπtitated in milligrams (mgj per ol and converted to % seasoning by comparison to a 6% equivalent seasoning control.

Kit Contents The Percent Seasoning kit comes packaged with all the reagents and membrane units needed to run the assay. The kit conlains Reagent A. Reagent B. packets to prepare Preparation reagent, one Calibrator Seπes. and one Control. Each kit comes with the appropriate number of disposable membrane holders and membrane units. The kit should be stored al 4*C.unlιl used and left out after first use for up to one month at room temperature.

Safety Precaution: Handle with care. The Percent Seasoning WwiB tain 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. Do not binel bv mouth. Oo not ingest, as one component is tonic.

Materials Required

Production Package: SafTesf Workstation: Disoosabfe Labware: Two Bottle-Top Dispensers SafTesf Analyzer Kimwipes One Reagent Holder SafTesf Filtration Unit 12 mm Glass Test Tubes

Test Tube Caps

Adjustable Pipette Pipette Tips 13 x 7 5mm Test Tubes 100ml bottles and caps Mute Display Toner Plastic baggies

Reagent/ Dispenser Preparation

NOTE: Bung reagents and dispensers tn room temperature (1β-2S*C) before beginning assay.

1. Mx the contents of the reagent bottles by gently swirling prior to attaching dupensers.

2. Attach the dispenser labeled "Seasoning A" onto the reagent bottle labeled "Percent Seasoning Reagent A' am) for "Seasoning B" onto Ihe reagent bottle labeled "Percent Seasoning Reagent B. The volumes are preset to the pecilied volumes in the Percent Seasoning Instruction section In the SafTest"' Binder.

3 To prepare Preparation Reagent add the contents of one packet marked "Preparation Reagent Salts" mto 5Uteπ ol distilled water.

4. Mia tor 5 minutes usmg a stir bar in a large beaker. Save for use in assays throughout the day.

5. Dispense approximately 1 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 Ihe particular seasoning being tested. Prepare andVor dilute the chip sample appropriately, according to the sample preparation section in the SafTest"' Quick Start Card.

Pre-Assay Preparation

1. At the first assay each day. label a new set of test tubes: one lor each of the calibrators, one tube for Ihe control, and 1.2, 3. etc. for the samples. II running replicates, label the appropnate amount of tubes for each control and sample.

2. Using the adjustable pipette, traπslβr the appropnate volume of each calibrator, control, and sample into the designated test tubes. Refer to the Percent Seasoning instruction section in the SafTest" Binder lor aliquoting volumes Wipe the pipette p prior to dispensing all samples, controls, and calibrators. Use a new tip lor each sample, reagent blank, control, and calϊirator.

3. After the first calibration 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.

Test Method] 1. For Ihβ first assay of the day turn on the SafTesf" Analyzer for 5 minutes Place the 570/S90 filter in Ihe filter compartment Select the "SEA FL" program.

2. Dispense one aliquot of Percent Seasoning Reagent A into each of the calibrator tubes, the control tube, and each of the sample tubes. Dispense one aliquot of Percent Seasoning Reagent B into each of the calibrator lubes, the centred tube, and each of Ihe sample tubes.

3. Cap an the tubes and invert gently 3-5 times to mx.

4. Set timer for 2 minutes. For the first assay turn on the SafTesf Analyzer for 5 minutes. Place the 5707690 filter m the filter compartment. Select the "SEA FL" program.

5. Zero the instrument by Inserting a tube containing water blank. Calibrate the Instrument by inserting the calibrators *! the proper order as prompted by the SafTest"" analyzer.

6. Following successful calibration, insert the control tube. The control should be run every time the samples are run. To ensure the instrument and reagents are performing properly, the control value should fall within the range staled on the package insert for the lot of controls used (see Package Insert - Control for Percent Seasoning Kits). II the control fans ignificantly outside the range, rerun the assay.

7. If Ihe control value falls within the range, insert the sample tubes in the designated order to analyze in the SafTest" Analyzer. Wipe each test tube with a rmt-liβe tissue prior to insertion in the SafTeat^m Analyzer. β. Al the end of the day. store calibrators, control m the refrigerator, and reagent bottles with dispensers attached at room temperature

Reporting Results

The SafTest"* Analyzer will use the calibrators to calculate Ihe protein concentration in mg/dL of controls and the solubilized chip.

II Ihe sample value is greater than Ihe value of the highest calibrator, the instrument win flag the result as Ηl.' The sample must be diluted at a higher dilution and retested. Values that are flagged W are inaccurate and should not be reported.

Check the instrument printout for nags or error messages before reporting results.

For laded curve fit (r* < 0.990), repeat Ihβ assay.

The range for the control is found on the package insert provided with the Percent Seasoning Control. The assay value lor the control should approximate this range.

Limitations New food matrices with no established protocols are considered special applications and should be cheeked for interference and spike recovery Refer to the Sample Preparation table found in the SafTest"' Binder.

Applications For special applications to this assay, call the SAI Product Support Number f -880-321 -SAFE.

Claims

What 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.