Title of Invention

A VERTICLE FLOW TEST STRIP FOR USE IN THE DIRECT DETECTION OF CHOLESTEROL PRODUCED FROM LOW DENSITY LIPOPROTEINS IN A WHOLE BLOOD, PLASMA OR SERUM SAMPLE

Abstract Cholesterol from Low Density Lipoproteins(LDL-C) is measured directly with a test strip at room temperature using a reagent that takes advantage of the varying surface charge density on LDLs and non-LDLs to selectively make LDLC available for testing.
Full Text A VERTICLE FLOW TEST STRIP FOR USE IN THE DIRECT DETECTION OF CHOLESTEROL PRODUCED FROM LOW DENSITY LIPOPROTEINS IN A WHOLE BLOOD, PLASMA OR SERUM SAMPLE
Background of the Invention
[001] This invention relates generally to the in vitro analysis, using a dry test strip, of plasma, serum or whole blood samples, and more specifically, to assay for cholesterol from Low Density Lipoproteins (LDL-C) containec in samples.
[002] The level of cholesterol in blood nas become accepted as a significant indicator of risk of coronary heart disease. Cholesterol is conta'ned and is transported in lipoproteins in blood. "Total Cholesterol" includes cholesterol from Low Density Lipoproteins (LDL-C), from Intermediate Density Lipoproteins (IDL-C), from Chylomicrons, from Very Lew Density Lipoproteins (VLDL-C) and from high density lipoproteins (HDL-C). It is well established from epidemiological and clinical studies that there is a positive correlation oetween levels of LDL-C arid to a lesser extent of Lp(a)-C to coronary heart disease. Traa:tionally, LDL-C has been identified as "bad" cholesterol". On the otner hand, clinical studies have established a negative correlation between levels of HDL-C ("good" cholesterol) ana coronary heart disease. Standing alone, the level of total cholesterol in blood, which is a measure o:* the sum total of HDL-C, LDL-C. IDL-C, VLDL-C and Chylomicrons-C, is not generally regarded as an adequate indicator of the risk of coronary heart disease because tne overall level of total cholesterol does not reveal the relative proportions of choiesteroi r'rcm these

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sources. To better assess the risk of heart disease, it is desirable to determine the amount of LDL-C in a sample in addition to the total cholesterol in the sample.
[003] The most common approach to determining LDL-C in the clinical laboratory is the Friedewald calculation, which estimates LDL-C from measurements of total cholesterol, HDL-C and triglycarides. Although convenient, the Friedewald calculation suffers from several weil-established drawbacks. Nauck et al. "Methods for Measurement of LDL-Cholesteroi: A Critical Assessment of Direct Measurement by Homogeneous Assays versus Calculation" Clin. Chem. 48.2 (2002). For example, because the Friedewald calculation involves measurements other than LDL-C, it is subject to potential' compounded inaccuracies from the determinations of the other lipids in the equation. Further, its usefulness is known to be limited to biological fluids with triglyceride levels below 400 mg/dL, and its accuracy reportedly declines with tnglyceride levels greater than 200 mg/dL.
[004] Uitra-centnfugation is a known technique to separate and to quantify the various lipoprotein components from serum or plasma samples. However, ultra-centrifugation is tedious, time consuming, and the highly labile lipoproteins can be substantially altered by the high salt concentrations that are a part of the ultra-centrifugation process as well as by centrifugal forces. "Furthermore, a plethora of different types of equipment and tubes are used, making conditions
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difficult to reproduce from one laboratory to another and consistent separations highly dependent en the skilis and care of the technician. Id_ At 238.
[005] Another technique for measuring LDL-C is electrophoresis This technique also has certain drawbacks. Electrophoresis gel assays ao not lend themselves readily to automation and their accuracy and repeatac.iity depends at least in part on the technique of the technician performing the test.
[006] Other so-called homogeneous methods that involve precipitation of non-LDL lipoproteins, heating and additional steps, have recently become available. One. homogeneous method for determining LDL-C is disclosed in U S. Patent No. 5,888,827 (Kayahara, Sugiuchi, et al.; assigned to Kyowa Medex Co., Japan). The '827 patent describes a two-stage liquid phase reaction to quantify LDL-C concentration in a fluid sample. In the first step, the sample containing LDL-C is placed in a first reagent that includes tnmethyl Deta-cyclodextrin as a sugar compound, pciycxyethylene monclaurate as a protein sol^bilizing agent, EMSE (N-ethyl-N-(3-methylphenyl)-N',succinylethylenediamene) and Tris buffer. The reaction mixture is men heated to 37°C, and after 5 minutes the absorbance is read. A second reagent including cholesterol esterase, cholesterol oxidase, peroxidase, 4-aminoantipyrine and Tris buffer is then added and after another 5 minutes the absorbance is again measured at the same wavelength LDL-C is then calculated by separately subjecting a standard solution of cholesterol to the same procedure ana comparing the respective absorbance values For many applications the manipulations required in the practice of this method such as
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heating, multiple reagents and multiple readings is considered a drawback. Because this method is complex and tedious to perfo'-m even in a laboratory, it would not be suitable for a point-of-care (POC) environment.
[007] Another two-stage homogeneous assay is disclosed in U.S. Patent No. 6,194,164 (Matsui et al.; assigned to Denke Seiken, Ltd. Japan). In the first stage, HDL-C, VLDL-C and Chylomicron-C in the test sample are eliminated and, in the second step, the cholesterol remaining in the test sample (viz., LDL) is quantified. In the first step, cholesterol esterase and cholesterol oxidase act on the test sample in the presence of a surfactant that acts on lipoprotems other than LDL-C ("non-LDLs"). The hydrogen peroxide thereby generated is decomposed to water and oxygen by catalase. Alternatively, a phenol-based or aniline-based hydrogen, donor is reacted with the hydrogen peroxide to convert it to a colorless compound. Preferred surfactants that act on the non-LDLs include polyoxyethylene laurl ether, polyoxyethylere cety! ether, polyoxyethylene oleyl ether, poiyoxyethylene higher alcohol ether, and the like. In the second reaction disclosed in the '164 patent, cholesterol remaining in the test sample, which should theoretically contain only LDL-C, is quantified. Tne second step may be carried out by adding a surfactant that acts on at least LDL and quantifying the resulting hydrogen peroxide by the action of the cholesterol esterase and the cholesterol oxidase added in the first step.
[008] As with the metnod disclosed in the '827 patent, one disadvantage of the method taught by the '164 patent is that it requires heating the reaction
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mixture to a temperature of 37° C, and experimental data indicates that tne test accuracy suffers at lower temperatures. Also as taught in the '827 patent, the method of the '164 patent requires multiple reagents to be added at different times, making it equally sncompatibie with POC testing or use in ove'-tne counter ("OTC") applications.
[009] A homogeneous assay for measuring LDL-C in serum was disclosed by H. Sugiuchi et al., Clinical Chemistry 44:3 522-531 (1998). Tnis disclosure shows a correlation between the use.of a combination of tnblocK copolymer and alpha-cyclodextrin sulfate and the selective enzymatic reaction of LDL-C when both LDLs and non-LDLs are contacted with the combination in a liquid assay system. The preferred polyoxyethylene-polyoxypropylene block copolymer of the Sugiuchi et al. disclosure exhibited limited solubility under liquid assay reaction conditions, rendenng the adaptation to a dry strip unworkable
[010] Co-pending and commonly assigned U.S. patent application 10/663,555, filed September 16. 2C03, discloses a one-step, room-temperature whole blood, dry chemistry assay for LDL-C in which the amount of LDL-C present in whole blood is calculated from the results of direct measurements of total cnoiesterol and non-LDL-C. Although the disclosed assay overcomes most of the problems of the multi-step, wet chemistry LDL cholesterol assays of the prior art, there remains a preference for direct assays. Thus there remains a need for a convenient, easy to use, dry, one-step, room-temperature diagnostic test for directly measuring LDL-C.
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Summary of the Invention
[011] These and other problems of prior art assays for LDL-C are overcome by the present invention. The present invention, in one aspect is a direct, room-temperature method for the detection and measurement of cholesterol from low-density lipoproteins in a plasma, serum or whole blood sample. The method comprises treating a sample that includes both LDLs and non-LDLs so that enzymatic conversion of LDL-C is encouraged while enzymatic turnover of non-LDL-C is retarded or blocked. The sample is treated by contacting it with a combination of reagents that relate to LDLs and to non-LDLs differently as a function of their differing surface charge density. Any reagents that correspond with the various lipoproteins in a sample as a function of surface charge density carried by the lipoproteins in such a way that selectively encourages enzymatic conversion of cholesterol carried by LDLs while blocking or retarding such conversion in the other types of lipoprotein cholesterol present may be usec
[012] This invention is based in part on the discovery that the differing surface charge density of the LDLs and non-LDLs in a sample can be used to advantage. The sparsely negatively charged surface characteristics, measured at or near physiological pH, of chylomicron, VLDLs, and IDLs cause them to bind to certain anionic polymers and, in particular, sulfates. Although good results have been observed in connection with a range of dextran sulfates, a polyanion, having molecular weights from about 5,000 to about 50,000, the best results to
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date have been obtained when these polyanions are used in conjunction with alpha cyclodextnn sulfate or other cyclodextrin derivatives.
[013] HDL's are found to be generally strongly negatively charged and have been found to be blocked from producing cholesterol, or temporarily protected from the activity of cholesterol-producing enzymes, when bound with specific combinations of such sulfates and with a copolymenc surfactant. Although simple polypropyiene glycol and or polyethyiene glycol molecules are found to also inhibit the enzymatic conversion of HDL-C, the preferred copolymenc surfactant is a polyoxyethyiene-polyoxypropylene-poiyoxyethylene hybrid, having a molecular weight range from about 2,100 to about 6,000 with a preponderance of polyoxyproylene. Preferably the polyoxypropylene comprises 8C-95% of the copolymer surfactant.
[014] Another aspect of the invention is based in part on the discovery that certain lower molecular weight surfactants can be used to increase the solubility of high molecular weight block copolymer surfactants, making them usefui in test strip assays for direct measurement of LDL-C. In the present invention, the limited solubility of these preferred compounds has been addressed by the use of a surfactant system that in part functions on three different leveis. In the first level, the surfactants of the present invention will aid to solubiiize the polyoxyethylene-polyoxypropylene-polyoxyethylene hybrid without diminished selectivity in enzymatic conversion of the LDL-C relative to the ron-LDL-C analytes in a sample. The second level of surfactants in pan produce mixed
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micelles that, in a multi-membrane or multi-layer test strip, transport the tnblock copolymer and released LDL-C from a reagent-containing membrane to a cholesterol-reaction membrane. The third level of surfactants, wmch in practice are normally directly adjacent or impregnated on a cholesterol reaction membrane in a test strip, function in part to solubilize or emulsify released cholesterol from the mixed micelies, containing the triolocK copolymer and other surfactants, so that the cholesterol can react with the enzyme system of the cholesterol reaction membrane.
[015] Selective treatment of non-LDLs in a sample by such reagents is enabled by the use of a cationic species connecting them selectively to non-LDLs. In one aspect of the invention, the cationic species is a divalent metal bridge. The divalent metal bridge has been observed to link the reagents to the surfaces of non-LDLs, which have a sufficiently dense negative surface charge that the surface charge of the LDLs in the same sample is relatively siightly positive. Although good results have been achieved with magnesium, other divalent metals such as calcium, manganese and others coulc be used. In addition, any materials that can electrostatically bond to the negatively charged surface of the lipoprotein structures and/or the poiyanion can exhibit similar enzymatic selectivity. As an example, good results have been achieved using triethanolamine hydrochloride as the cationic species for the bridging component.
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in the sample and that work together to block or retard the production of cholesterol from non-LDLs while facilitating the production of cholesterol frorr, LDLs. The supply of such materials is normally deposited on one or more layers in the vertical flow path of the blood sample so that the materials are brought into solution following separation of red blood cells from the sample. However, the supply of materials could also be located prior to the red blood cell separation mechanism.
[020] Tne materials are selected to work with the eiectrical characteristics of the non-LDL components sought to be b.ocked from the production of cholesterol. Typically, the materials include a divalent meta! ion source capable of forming a bridge between the electrically negative components, isted above, while avoiding the formation of such a bridge between LDL in the sample and the protective components due to the diminished anionic electrical surface characteristics normally found in the LDLs.
[021] The test strip also includes, in the flow path of the blood sample and furthest away from the application point, a supply of materials selected to resuit in a detectible color change following enzymatic conversion of the produced cholesterol.
[022] It is one general object of the invention to provide a dry phase test strip chemistry for testing the concentration of analytes in a body fluid A more specific object is to provide a dry test strip capable of directly determining the concentration of LDL-C in whole blood or plasma.
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[023] One significant benefit of the present invention is that the LDL-C concentration can be directly determined in a single stage assay. Another benefit is that the diagnostic test can be performed at room-temperature. Other benefits and objects of the invention will be discerned upon consideration of the following description of the invention.
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Brief Description Of The Drawings [024] FIG. 1 shows a test strip according to the present invention.
[025] FIG. 2 shows the correlation between LDL-C as determined by gel electrophoresis and a measured %R ootamed by dry test strips prepared according to one embodiment of the present invention identified as Example 1.
[026] FIGS. 3-9 show the correlation between LDL-C as determined by gel electrophoresis and a measured %R obtained by dry test strips prepared according other embodiments of the present invention identified as Exampies 9-
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Description of the Preferred Embodiments
[027] For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings and described in the following written specification. It is understood that no limitation to the scope of the invention is thereby intended. It is further understood that the present invention includes any alterations and modifications to the illustrated embodiments and includes further applications of the principles of the invention as would normally occur to one sKilled in the art to which this invention pertains.
[028] One useful embodiment of the present invention is shown in FIG. 1. Elements or layers M-1, M-2, M-3, M-4 and M-5 are held between sampie application port 1 and read port 4 and define the vertical pathway traversed by the sample following the application of serum, plasma or whole blood to at sample application port 1.
[029] In this embodiment, the sampie first may encounter an optional spreading layer that is not shown in FIG. 1 but would be directly above Layer M-1. The purpose of spreading layer, if present, is to spread the sample relatively evenly over an area of port 1 that is larger than the application point. In addition, the spreading layer may be impregnated with the above-descriDed reagents. One purpose of impregnation of the spreading layer, if it is present, is to orovide a longer contact time between the applied sample and the reagents.
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[030] Blood separation layer M-1 is, in this embodiment, at least a part of the mechanism for blocking or retarding the flow of red blood cells. In a specific example, layer M-1 is a non-woven giass fiber layer available from Ahlstrorn Corporation, under the trade name "TuffGlass 144". Layer M-1 may contain dextran sulfate, a divalent metal or equivalent, cyclodextrin molecules, buffers, solubilizers such as sorbitol or sucrose and surfactants, including but not limited to the copolymer or triblock polymer surfactants that exhibit LDL or non-LDL selectivity.
[031] Like Layer M-1, Layer M-2 can also function to limit or retard the movement of red blood cells through the test strip and corresponding membranes. M-2 is typically an asymmetric polysulfone membrane witn a high degree of asymmetry. In the preferred embodimen: of this invention, the membrane is BTS SP-300 available from Pall Life Sciences. This layer may also contain each of the elements descriDed in M-1 with the addition of reagents in concentrations that are markedly different than M-1. In addition, surfactants may be present to increase the mobility of cholesterol released from the lipoprotein structures. Specifically, M-2 may contain all or part of the polyanion such as the dextran sulfate, a divalent or other cationic species required for prooer blocking of non-LDLs, all or part of the cyclodextrin molecules, surfactants, and in particular all or part of the ccpolymer or triblock polymer utiiizeo to block HDL-C and/or make LDL-C available. As with the layer M-1 layer M-2 may also include solubilizers, such as Sorbitol and sucrose.
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[032] The supply of divalent metal or other cationic species car, or'ginate from either M-1. M-2 or the spreading mesh, although the preferred location is M-2 or additionally M-4. The divalent metal may be, for example calcium, magnesium or manganese. The most preferred cation is magnesium which was chosen for its low cost, availability and ease of handling The cat;cr may also be a positively charged amine capable of binding lipoproteins One preferred amme is a tertiary amine such as tnethanolamr.e.
[033] In the embodiment shown in FIG. 1, layer M-2 is also a blood separation layer. It is an asymmetric material with a pore size of 300 microns on the sample-receiving side and about three (3) microns on the detection side. In addition to helping block or retard the 'low of red blood cells, it also slows the flow of the entire blood sample along the vertical path to increase the contact time of the sample with the reagents.
[034] Like M-2, the element identified in FIG. 1 as M-3 is a membrane that slows the rate of flow of the applied sample through the vertical arrangement so as to increase the amount of time the sample is in contact with the reagent membranes although this membrane rarely is treated with reagents designed to impart lipoprotem selectivity. The design objective for M-3 is controlled hydrophilicity and pore size to attenuate flow of the sample material through the test strip. A number of different memoranes have been effective to this end although the membrane of choice is a nydrophilic polyether sulfone with a trade name of Supor 1200 available from Pall Life Sciences. Also especially effective
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membranes of element M-3 are track-etched polycarbonate membranes such Poretics 0.4 Micron from Osmonics Inc. In most cases, this membrane is untreated except for surfactants or other wetting agents that may facilitate the spreading of the sample across the membrane surface
[035] The element designated in FIG. 1 as M-4 is aisc a reagent membrane layer and can optionally contain the same reagents as M-2 although in different proportions. Like M-2 the preferred membrane is an asymmetric polysulfone like BTS SP-300 available from Pal! Life Sciences. In some examples of the present invention, M-4 can be optional depending at least in part on the composition, reagents and arrangements of the elements M-1, M-2, M-3 and the optional spreading mesh not illustrated in FIG. 1.
[036] The layer illustrated as M-5 in FIG. 1 is the cholesterol detection membrane, which may be the membrane described in the co-pending and commonly assigned U.S. patent application 10/663,555, filed September 16, 2003
Example 1
[037] A dry strip was constructed based on the following membranes anc arrangement relative to FIG. 1:
Layer M-1; Tuffglass impregnated as describee in "Part A". Layer M-2; BTS-300 impregnated as described in "Part B". Layer M-3; Supor 1200, untreated
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Layer M-4; BTS-300 impregnated as described in "Part B'.
Layer M-5; Biodync A. as described in. the co-pending and commonly assigned U.S. patent application ".0/663,555, filed September 16, 20C3
[038] M-1. Tuffglass was dipped in solution "Part A" ana was dr.ed with moving air at 38°F ±2.5°C.
Part A
[039] To 300 mL of laboratory D.I. water the following was added. MES buffer 3.50 g, Sorbitol 9.0 g, sucrose, S.C g. polyethylene glycol 200 mwt 7.0 g dextran sulfate 10K mwi. 10.03g. NaCi 2.01 g. The pH of the solution was adjusted to a pH of 5.90 +/- 0.1 with 5 N NaOH. A total of 2 80 mL of 5 N NaOH was added to give a final pH was 5.85.
[040] From this stock solution, 169.89 grams were removed and placed in a 250 mL beaker. To this beaker, 2.0 g of dextran sulfate 10K mwt were dissolved. The pH was adjusted with 760 uL5 N NaOH to give a final pH of
5.95. The Tuffglass was dipped into this solution and was hung vertically to allow the excess solution to drip off the membrane. The membrane was then placed in the clipboard and dried horizontally in the dy;ng tunnel using standard heated conditions.
PartB
[041] To 200.15 g laboratory D.i water the following was added in order MES buffer 2.0 g, Sorbitol 9.06 g MgCI26H20 7.04 g. The pH was adjusted
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6.03 with 1.025 mL of 5 N NaOH. The solution was then chilled to 5°C followed by the addition of the following: co-cyciodextrin sulfate1.38 g, Siiwet L-77 0.73 g,
Pluronic L121 1.66g, Pluronic L43 0.45g. The solution was kept chilled during all additions. The Pyrex glass dish used to dip the membrane was chilled in the freezer before the addition of the impregnation reagent mixture. Approximately 70 mL of the "Part B" solution was added to the chilled glass vessel. The membranes were dipped and hung vertically for drying. Excess reagent was allowed to drip from the membrane that was dried without heat or application of moving air.
[042] FIG. 2 illustrates the data generated from the construction of the strip of Example 1 using twelve different blood samples with the results of each sample being an average of six strip results. Control aliquots of the same sample were tested for LDL-C by gel electrophoresis. The correlation between these control aliquots and the assays performed according to the method and device of the present invention was found to be good, as shown in FIG. 2.
Example 2
[043] A dry strip was constructed based on the following membranes and arrangement relative to FIG. 1:
Layer M-1; Tuff Glass impregnated as described in "Part C".
Layer M-2; Not present.
Layer M-3; Supor 1200, untreated.
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Layer M-4; BTS SP300 impregnated as described in "Part D". Layer M-5; Bioc'yne A.
PartC
[044] The following solution was impregnated onto a depth filter, which can encompass an amorphous fiber or a composite material of either glass, polymer or a random composite matrix. The impregnation can be by any known methods, such as dipping, spraying or freeze drying to produce the top reagent layer of the dry strip.
[045] To 50 mL of D. I. water the following were added: 1.23g of MOPS buffer, 1.5g dextran sulfate with an average molecular weight of 10,000, 0.5g a-cyclodextrin sulfate, 2.99g Sorbitol, 3.0g sucrose, and 0.6g magnesium chloride, all in 50ml of D.I. water. The pH was adjusted to 7.17 using 1ml of 5N NaOH.
PartD
[046] The following solution was impregnated onto a membrane that can be in part also utilized to separate red blood cells from a whole blood sample to yield either plasma or serum to the detection layer M-5 as well as to control reagent reconstitution either in the presently treated membrane or a subsequent reagent treated membrane or other substrate.
[047] To 300 mL of D.I. water, the following reagents were added: 6.01 g Pluronic L121, 4.32g magnesium chloride, 3.0g MOPS buffer, 4.13g alpha-cyclodextrin suifate, 0.63g MOPS buffer, 1.08g Sorbitol, 1.11g sucrose, 0.47mg Silwet L-77. The pH of the solution was 6.95 unaltered. The cloud point of the
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solution was 20°C. The layer was treated with 60.09g of this solution.
Example 3
[048] A dry strip was constructed based on the following membranes and arrangement relative to FIG. 1:
Layer M-1; Tuff Glass impregnated as described in "Part E".
Layer M-2; BTS SP300 impregnated as described in "Part F".
Layer M-3; Supor 1200, untreated.
Layer M-4; Not present.
Layer M-5; Biodyne A.
PartE
[049] To 50 mL of D.I. water the following were added: 1,2g of MOPS buffer, 2.5g dextran sulfate 10K, 0.5g alpha-cyclodextrin suifate, 2.01 g Sorbitol, 2.0g sucrose, and 0.6g magnesium chloride. The pH was adjusted to 7.16 using 1ml of 5N NaOH.
Part F
[050] To 300 mL of D.I. water, the following reagents were added: 6.19g Pluronic L121, 3.22g magnesium chloride, 3.0g MOPS buffer, 4.0g alpha-cyclodextrin sulfate, 0.55g MOPS buffer, 1.1g Sorbitol, 1.12g sucrose, 1.88g Silwet L-77, 1.05g Pluronic L121. The final pH of tha unaltered solution was 7.0. The layer was treated with 60.09g of this solution.
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Example 4
[051] A dry strip for this example was constructed with the same membranes as in Example 3, namely Tuff Glass (M-1), BTS SP300 (M-2), Supor 1200 (M-3), and Cholesterol reaction membrane (M-5).
[052] The Tuff Glass layer (M-1) was treated with 4.32g of MOPS buffer, 8.87g dextran sulfate 10K, 0.5g alpha-cyciodextrin sulfate, 9.9g Sorbitol, 11.25g sucrose, and 2.28g magnesium chloride, 7.4g Polyethylene Glycol, in 168.33g of deionized water. The pH was adjusted to 7.11 using 0.4ml of 5N NaOH.
[053] The BTS SP300 layer (M-2) was treated with 30.02g of the following solution: 5.42g Pluronic L121, 7.05g magnesium chloride, 2.0g MOPS buffer, 4.592g a-cyclodextrin sulfate, 9.01g Sorbitol, 0.75g hydroxypropyl cellulose, 1.38g dextran sulfate 10K, 2.47g Silwet L-77 in 100ml of deionized water, to which was added 0.33g MOPS buffer, 0.65g Sorbitol, 0.67g sucrose, ~29mg Siiwet L-77, 0.09g Tetronic 1107. The final pH of the solution was 7.27 with 0.1ml of 5N NaOH. There was no treatment to the Supor 1200.
Example 5
[054] A dry strip for this example was constructed with the same membranes as in Example 3, namely Tuff Glass (M-1), BTS SP300 (M-2), Supor 1200 (M-3), and Cholesterol reaction membrane (M-5).
[055] The Tuff Glass layer (M-1), was treated with 0.35g Pluronic L121, 0.06g Tetronic 304, 1.56g of MES buffer, 3.11g dextran sulfate 10K, 0.7687g
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alpha-cyclodextrin sulfate, 2.51 g Sorbitol, 1.17g sucrose, and 1.1g magnesium chloride, 0.1ml Silwet L-77, in 75.0g of deionized water. The pH was adjusted to 6.14 using 0.4ml of 5N NaOH.
[056] The BTS SP300 (M-2), was treated with 1.80g Pluronic L121, 0.91g dextran sulfate 10K, 0.7477g alpha-cyclodextrin sulfate, 0.8g MOPS buffer, 2.0g Sorbitol, 0.61g sucrose, 0.9g magnesium chloride, 0.29g Tetronic 1107 all in 75g of deionized water. The final pH of the solution was 7.17 with 0.15ml of 5N NaOH. There was no treatment to the Supor 1200.
Example 6
[057] A dry strip was constructed based on the following membranes and arrangement relative to FIG. 1:
Layer M-1; Tuff Glass impregnated as described in "Part G",
subsequently impregnated as described in "Part H", and subsequently treated as described in "Part I".
Layer M-2; BTS SP300 impregnated as described in "Part J"
subsequently impregnated as described in "Part K", and subsequently treated as described in "Part I".
Layer M-3; Supor 1200, untreated.
Layer M-4; Not present. Layer M-5; Biodyne A.
PartG
[058] To 1875,0g of D.I. water the following were added: 8.95g Pluronic L121, 17.85g Tetronic 304, 39.1g of MES buffer, 77.54g dextran sulfate 10K,
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19.2g alpha-cyclodextrin sulfate, 62.5g Sorbitol, 29.11 g sucrose, and 27.35g magnesium chloride, 2.5g Silwet L-77. The pH was adjusted to 6.14 using 0.4ml of 5N NaOH.
PartH
[059] The following solution was used to treat the membrane impregnated with PartG. To 199.6g of D.I. water the following were added: 8.16g dextran sulfate 10K, 1.41g alpha-cyclodextrin sulfate, 1.85g magnesium chloride, 3.45g MES buffer, 3.14g Sorbitol. The pH was adjusted to 6.24 using 1.4ml 5N NaOH.
Parti
[060] A 2.0 % polyvinyl alcohol solution was prepared to subsequently treat both Layer M-1 and Layer M-2,
Part J
[061] To 749.8 g of D.I. water, the following chemicals were added: 16.1 g Pluronic L121, 9.0g dextran sulfate 10K, 5.0g alpha-cyclodextrin sulfate, 7.9g MOPS buffer, 12.8g Sorbitol, 4.7g sucrose, 7.0g magnesium chloride, 3.42g Tetronic 1107, 2.2g Silwet L-77. The final pH of the solution was 7.22 with 3.0ml of5N NaOH.
PartK
[062] To 100 g of D.I. water the following chemicals were added. 1.5 Silwet L-77g, 1.05 Pluronic L121.
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Example 7
[063] Dry test strips were composed of the same membranes as in Example 3, namely: Tuff Glass (M-1), BTS SP300 (M-2), Supor 1200 (M-3), and Cholesterol reaction membrane (M-5). The Tuff Glass layer (M-1) was treated with 64.6g Pluronic L121, 5.79g Tetronic 304, 12.58g of MES buffer, 24.97g dextran suifate 10K, 6.16g alpha-cyclodextrin suifate, 20.Og Sorbitol, 9.3g sucrose, and 8.77g magnesium chloride, 0.79g Silwet L-77, in 599.63g of deionized water. The pH was adjusted to 6.21 using 5.5ml 5N NaOH.
[064] The BTS SP300 (M-2) in this example was treated with 3.6g Pluronic L121, 2.02g dextran suifate 10K, 1.53g alpha-cyclodextrin suifate, 1.78g MOPS buffer, 1.21g Sorbitol, 1.29g sucrose, 1.81g magnesium chloride, 0.62g Tetronic 1107, 1.03g Emulgen 21 OP, 1.51g hydroxypropyl (3-cyclodextrin all in 201.5g deionized water. Both of these membranes (M-1 and M-2) were run through a drying tunnel. There was no treatment to the Supor 1200.
Example 8
[065] Dry test strips were composed of the same membranes as in Example 3, namely: Tuff Glass (M-1), BTS SP300 (M-2), Supor 1200 (M-3), and Cholesterol reaction membrane (M-5). The Tuff Glass layer (M-1) was treated with 0.35g Pluronic L121, 0.06g Tetronic 304, 1.56g of MES buffer, 3.11g dextran suifate 10K, 0.7687g alpha-cyclodextrin suifate, 2.51 g Sorbitol, 1.17g sucrose, and 1.1g magnesium chloride, 0.1m! Silwet L-77, in 75.Og of deionized
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water. The pH was adjusted to 6.14 using 0.4ml of 5N NaOH.
[066] The BTS SP300 (M-2) was treated with 1.80g Pluronic L121, 0.91 g dextran sulfate 10K, 0.7477g alpha-cyclodextrin sulfate, 0.8g MOPS buffer, 2.0g Sorbitol, 0.61g sucrose, 0.9g magnesium chloride, 0.29g Tetronic 1107 all in 75g of deionized water. The final pH of the solution was 7.17 with 0.15ml of 5N NaOH. There was no treatment to the Supor 1200.
Example 9
[067] The dry strips of this example were composed of a non-glass fiber top layer (M-1), namely Accuwick Ultra, followed by a BTS SP300 layer (M-2), a BTS SP300 layer(M-4), and Cholesterol Detection Membrane (M-5). The Accuwick Ultra layer was treated with a solution of the following chemicals dissolved into 375g of deionized water: 7.80g of MES buffer, 15.57g dextran sulfate 10,000 mwt, 3.85g a-cyclodextrin sulfate, 12.5g D-Sorbitol, 5.82g sucrose, 5.47g magnesium chloride, 1.79g of Pluronic L121, 3.59g Tetronic 304, and 0.5g of Silwet L-77. The pH was adjusted to 6.16 using 2ml of 5N NaOH.
[068] The first BTS SP300 layer (M-2) was impregnated by dipping and rolling away the excess the following solution: into 187.5g of deionized water the following chemicals were dissolved; 2.18 g PVA 30-70K mwt, 1.75g Tetronic 304, 4.02g MES buffer, 7.77g Dextralip 15, 1.96g a-cyclodextrin sulfate, 7.31g D-Sorbitol, 1.40g sucrose, 3.52g MgSO4, 2.5g polyethylene glycol 6,000 mwt, 57
25

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mg Antifoam C. The pH of the above solution was adjusted to 6.2/ with 1.5 ml of 5 N NaOH.
[069] The second BTS SP300 layer (Wl-4) was treated with a solution consisting of the following chemicals dissolved in two solutions. The first solution consisted of 20.35g of a 4% PVA 30-70K mwt solution and 30.55g of a solution containing the following chemicals dissolved into 50.01 g of deionized water: 2.048g PVA 30-70K mwt, 2.31 g Pluronic L121, 1.20g dextran sulfate 10,000 mwt, 1.25g magnesium sulfate, 1.31g Bis Tris buffer, 1.04g g a-cyclodextrin sulfate, 3.75g of D-Sorbitol, 0.0256g Siivvet L-77, and 0.03g of Tetronic 30, 0.47g of CHAPS. The pH of the solution was 6.48 after adding ~2.5ml of 3.25 N HCL
[070] The correlation between control aliquots and sixteen assays using the test strips of Example 9 was found to be good, as shown in FIG. 3.
Example 10
[071] The dry strips of this example were composed of Tuff Glass (M-1), BTS SP300 (M-2), Supor 1200 (M-3), and Cholesterol Detection Membrane (M-5). The Tuff Glass layer (M-1) was treated with a solution of the following chemicals dissolved into 300g of deionized water: 6.27g of MES buffer, 12.41g dextran sulfate 10K, 3.06g a-cyclodextrin sulfate, 10.01g D-Sorbitol, 4.65g sucrose, 4.37g magnesium sulfate, 1.43g of Pluronic L121, 2.90g Tetronic 304, and 0.4g of Silwet L-77. The pH was adjusted to 6.15 using 1.8ml of 5N NaOH. The BTS
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SP300 was treated with a solution of the following chemicals dissolved into 296.5g of deionized water: 7.20g Piuronic L121, 3.6g dextran sulfate 10K, 3.58g magnesium sulfate, 3.15g MOPS buffer, 3.20g a-cyclodextrin sulfate, 8.13g of D-Sorbitol, 2.38g sucrose, and 1.2g Tetronic 304. The pH of the solution was 7.12 after adding 1ml of 5N NaOH. There was no treatment to the Supor 1200.
[072] The correlation between control aliquots and fourteen assays using the test strips of Example 10 was found to be good, as shown in FIG. 4. '
Example 11
[073] The dry strips of this example were composed of Tuff Glass (M-1), BTS SP300 (M-2), Supor 1200 (M-3), and Cholesterol Detection Membrane (M-5). The Tuff Glass layer (M-1) was treated with a solution of the following chemicals dissolved into 300g of deionized water: 6.67g of MES buffer, 12.57g dextran sulfate 10K, 3.07g a-cyclodextrin sulfate, 10.08g D-Sorbitol, 5.33g sucrose, 4.41g magnesium sulfate, 2.86g Tetronic 304, and 0.0710g of sodium azide. The pH was adjusted to 6.22 using 2.25 ml of 5N NaOH. This solution was applied to the membrane by dipping into the solution followed by rolling the excess off between two rollers, and allowed to air dry on an open fiber matrix.
[074] The BTS SP300 (M-2) was treated with a solution of the following chemicals dissolved into 500g of deionized water: 12g Piuronic L121, 5.99 dextran sulfate 10K, 5.99g magnesium sulfate, 5.18g MOPS buffer, 5.19g a-cyclodextrin sulfate, 4.01 g of D-Sorbitol, 4.01g sucrose, and 1.9g Tetronic 304.
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The pH of the solution was 7.19 after adding 1.5ml 01 5N NaOH. Lastly, the BTS SP300 was then sprayed with a treatment of 4.03g dextran sulfate 10K, 0.6g of a-cyclodextrin sulfate, 0.57g magnesium sulfate, 1.75g of MES buffer, and 2.0g D-Sorbitol dissolved into 100.1g of deionized water. The pH of the solution was 6.31 after adding 1.5ml of 5N NaOH. There was no treatment to the Supor 1200.
[075] The correlation between control aliquots and twenty-one assays using the test strips of Example 11 was found to be good, as shown in FIG. 5.
Example 12
1076] The dry strips of this example were composed of Tuff Glass (M-1), BTS SP300 (M-2), Supor 1200 (M-3), and Cholesterol Detection Membrane (M-5). The Tuff Glass layer (M-1) was treated with a solution of chemical reagents dissolved into 300 ml of deionized water: 6.67g of MES buffer, 12.57g dextran sulfate 10K, 3.07g a-cyclodextrin sulfate, 10.08g Sorbitol, 5.33g sucrose, 4.41 g magnesium sulfate, 2.86g Tetronic 304, and 0.0710g of sodium azide.. The pH was adjusted to 6.22 using 2.25ml of 5N NaOH.
[1077] The BTS SP300 (M-2) was treated with a solution resulting by dissolving the following chemicals into 500g of deionized water: 12g Pluronic L121, 5.99g magnesium sulfate, 5.18g MOPS buffer, 5.19g a-cyciodextrin suifate, 4.01 g of Sorbitol, 4.01 g sucrose, 5.99g dextran sulfate 10K and 1.9g Tetronic 304. The pH of the solution was 7.19 after adding 1.5ml of 5N NaOH.
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In addition, 0.50g of a solution containing the following: 9.99g Pluronic L123, 10.01g of Piuronic L101, 5.05g Pluronic L103, 9.99g Pluronic L61, 10.02 Pluronic L64, and 2.75g of Silwet L-77 were added to the BTS SP300 solution before impregnation. After the membrane was dried, it was then sprayed with the following chemicals dissolved intolOO g of D.I. water. 4.03 g of dextran sulfate 10Kmwt, 0.6 g a-cyclodextrin sulfate, 0.57g magnesium sulfate, 1.75g MES buffer and 2.0g D-Sorbitol. The pH of the solution was 6.31 after adding 1.5ml of 5N NaOH. Lastly, the BTS SP300 was then sprayed with a treatment consisting of 4.03g dextran suifate 10K, 0.6g of a-cyclodextrin sulfate, 0.57g magnesium sulfate, 1.75g of MES buffer, and 2.0g Sorbitol. The pH of the solution was 7.19 after adding 1.5ml of 5N NaOH. There was no treatment to the Supor 1200.
[078] The correlation between control aliquots and twenty-one assays using the test strips of Example 12 was found to be good, as shown in FIG. 6.
Example 13
[079] The dry strips of this example were composed of Tuff Glass (M-1), BTS SP300 (M-2), Supor 1200 (M-3), and Cholesterol Membrane (M-5). The Tuff Glass layer (M-1) was treated with 6.67g of MES buffer, 12.57g dextran sulfate 10K, 3.07g a-cyclodextrin sulfate, 10.08g Sorbitol, 5.33g sucrose, 4.41 g magnesium sulfate, 2.86g Tetronic 304, and 0.071 Og of sodium azide all in 300ml of deionized water. The pH was adjusted to 6.22 using 2.25ml of 5N
29

WO 2005/074609 PCT/US2005/003234
NaOH. After the membrane had dried, the Tuff Glass was then sprayed with a treatment of 4.03g dextran sulfate 10K, 0.6g of a-cyclodextrin sulfate, 0.57g magnesium sulfate, 1.75g of MES buffer, and 2.0g D-Sorbitol all in 100g of deionized water. The pH was adjusted to 6.31 with 1.5ml of 5N NaOH.
[080] The BTS SP300 (M-2) was treated with 18.8g Pluronic L121, 2.90g magnesium sulfate, 7.37g MOPS buffer, 8.96g a-cyclodextrin sulfate, 7.38g of Sorbitol, 6.00g sucrose, 10.11 dextran sulfate 10K, 7.12g Tetronic 304, 2.90g Silwet L-77 and 0.15g sodium azide, all dissolved in 749.5g of deionized water. The pH of the solution was 7.15 after adding 2.5ml of 5N NaOH. In addition, 1.50g of a the following solution was added to the above solution before impregnation: 9.99g Pluronic L123, 10.01g Piuronic L101, 5.05g Pluronic L103, 9.99g Pluronic L61, 10.02g Pluronic L64, and 2.75g Silwet L-77. After the membrane had dried, it was sprayed with the following chemicals dissolved into 100g of D.I. water: 4.03g dextran sulfate 10K mwt, 0.6g of a-cyclodextrin sulfate, 0.57g magnesium sulfate, 1.75g of MES buffer, and 2.0g Sorbiiol. There was no treatment to the Supor 1200.
[081] The correlation between control aliquots and fifteen assays using the test strips of Example 13 was found to be good, as shown in FIG. 7.
Example 14
[082] The dry strips of this example were composed of Tuff Glass (M-1), BTS SP300 (M-2), Supor 1200 (M-3), and Cholesterol Membrane (M-5). The Tuff Glass layer (M-1) was treated with 6.6g of MES buffer, 12.57g dextran sulfate
30

WO 2005/074609 PCT/US2005/003234
10K, 3.07g a-cyclodextrin sulfate, 10.08g Sorbitol, 5.33g sucrose, 4.41 g magnesium sulfate, 2.86g Tetronic 304, and 0.071 Og of sodium azide all in 300m! of deionized water. The pH was adjusted to 6.22 using 2.25ml of 5N NaOH. After the membrane had dried, the Tuff Glass was then sprayed with a treatment of 4.03g dextran sulfate 10K, 0.6g of a-cyclodextrin sulfate, 0.57g magnesium sulfate, 1.75g of MES buffer, and 2.0g Sorbitol dissolved in 100g of deionized water. The pH was adjusted to 6.31 with 1.50ml of 5N NaOH. The Tuff Glass layer was next sprayed with a 2% solution of PVA.
[083] The BTS SP300 (M-2) was treated with 18.8g Pluronic L121, 2.90g magnesium sulfate, 7.37g MOPS buffer, 8.96g a-cyclodextrin sulfate, 7.38g of Sorbitol, 6.00g sucrose, 10.11g dextran sulfate 10K, 2.90g Silwet L-77, 7.12g Tetronic 304, and 0.15g of sodium azide, all in 749.5g of deionized water. The pH of this solution was adjusted to 7.15 by 2.5ml of 5N NaOH. When dried, the BTS SP300 was then sprayed with a treatment of 24.00g dextran sulfate 10K, 3.57g of a-cyclodextrin sulfate, 3.58g magnesium sulfate, 10.78g of MES buffer, and 11.82g D-Sorbitol dissolved in 600g of deionized water. The pH was adjusted to 6.20 with 2.0ml of 5N NaOH. Lastly, the BTS SP300 (M-2) was then sprayed with a treatment of 0.15% Silwet L-77 and 1.0% Pluronic L121. There was no treatment to the Supor 1200.
[084] The correlation between control aiiquots and fourteen assays using the test strips of Example 14 was found to be good, as shown in FIG. 8.
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Example 15
[085] The dry strips of this example were composed of the non-glass fiber layer Accuwick Ultra (M-1), BTS SP300 (M-2), Supor 1200 (M-3), and Cholesterol Membrane (M-5). The Accuwick Ultra layer (M-1) was treated by dissolving the following chemicals into 300g of deionized water: 6.30g of MES buffer, 12.43g dextran sulfate 10K, 3.08g a-cyclodextrin sulfate, 10.04g Sorbitol, 4.63g sucrose, 4.37g magnesium sulfate, 2.86g Tetronic 304, 0.4g of Silwet L-77, and 1.47g of a solution containing the following' 1.03g (3-cyciodextrin polymer, 0.99g randomly methylated p -cyclodextrin. The layer was further treated with 2.98g of a solution containing the following: 2.99g Emulgen 21 OP, 9.00g Pluronic L121, 1.98g polypropylene glycol 3,500 mwt. The pH was adjusted to 6.22 using 1.75ml of 5N NaOH. There was no treatment to the Supor 1200.
[086] The BTS SP300 was treated with a solution resulting by dissolving the following chemicals into 300g deionized water: 5.43g Pluronic L121, 2.75g magnesium sulfate, 2.39g MOPS buffer, 2.39g a-cyclodextrin sulfate, 1.80g of Sorbitol, 1.82g sucrose, 1.50g Emulgen 21 OP, 0.45g of Tetronic 304, 0.47g Tetronic 150R1, 0.46g Tetronic 901, 2.33g hydroxypropyl (3-cyclodextrin. The pH of the solution was 7.21 after adding ~0.9ml of 5N NaOH.
[087] The correlation between control aliquots and fourteen assays using the test strips of Example 15 was found to be good, as shown in FIG. 9.
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[088] While the invention has been illustrated and described in detail in the drawings and foregoing description, the same should be considered as illustrative and not restrictive in character. It is understood that only the preferred embodiments have been presented and that all changes, modifications and further applications that come within the spirit of the invention are desired to be protected.
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WE CLAIM:
1. A vertical flow test strip for use in the direct detection of cholesterol produced from low density
lipoproteins in a whole blood, plasma or serum sample, the test strip comprising:
a) a red blood cell blocking membrane for blocking or slowing the progress of red blood
cells through the test strip;
b) a cholesterol detection membrane for providing a color change in the presence of
cholesterol; and
c) in said test strip between said red blood cell membrane and said cholesterol detection
membrane, a supply of a combination of reagents that can bind with non-LDL lipoproteins to block the
cholesterol in said non-LDL lipoproteins from being measured in said cholesterol detection membrane
while selectively permitting low density lipoprotein cholesterol (LDL-C) to be directly measured.
2. The test strip as claimed in claim 1, wherein said combination of reagents are selected from the
group consisting of cations, polyanions, cyclodextrin derivatives, a copolymeric surfactant and a
surfactant for the copolymeric surfactant.
3. The test strip as claimed in claim 2, wherein the cations include a divalent metal.
4. The test strip as claimed in claim 3, wherein the divalent metal is magnesium.
5. The test strip as claimed in claim 2, wherein the cations include a positively charged amine
effective to bind lipoproteins.
6. The test strip as claimed in claim 5, wherein the amine is triethanolamine hvdrochloride.
7. The test strip as claimed in claim 2, wherein the polyanion is dextran sulfate.
8. The test strip as claimed in claim 2, wherein the cyclodextrin derivative is alpha cyclodextrin
sulfate.
9. The test strip as claimed in claim 2, wherein the copolymeric surfactant is a polyoxyethylene-
polyoxypropylene-polyoxyethylene hybrid, having a molecular weight range from about 2,100 to about
6,000 with a preponderance of polyoxyethylene.
10. The test strip as claimed in claim 1, wherein the reagents include a high molecular weight
blocking copolymer surfactant effective to bind non-LDLs and a low molecular weight surfactant
effective to increase the solubility of the blocking copolymer surfactant.
11. The test strip as claimed in claim 1, wherein said red blood cell blocking membrane is
impregnated with at least some of the supply of a combination of reagents.
12. The test strip as claimed in claim 1, comprising at least one intermediate membrane
impregnated with at least some of the supply of a combination of reagents.
13. A vertical flow test strip for use in the direct detection of cholesterol produced from low density
lipoproteins in a whole blood, plasma or serum sample, the test strip comprising:
(a) a red blood cell blocking membrane for blocking or slowing the progress of red blood
cells through the test strip;
(b) a cholesterol detection membrane for providing a color change in the presence of
cholesterol; and
(c) a supply of a combination of reagents in said test strip that block the enzymatic conversion
of non-LDL lipoproteins and permitting the conversion of LDL-C lipoprotein cholesterol.
14. A test strip as claimed in claim 13, wherein said test strip functions at room temperature.
15. A test strip as claimed in claim 13, wherein said combination of reagents includes block
copolymer surfactants.
16. A test strip as claimed in claim 15, wherein said combination of reagents includes lower
molecular weight surfactants that increase the solubility of said block copolymer surfactants.
17. A test strip as claimed in claim 13, comprising triblock copolymer and surfactants impregnated
adjacent to said cholesterol detection membrane.
18. A test strip as claimed in claim 13, comprising triblock copolymer and surfactants impregnated
in said cholesterol detection membrane.


Cholesterol from Low Density Lipoproteins(LDL-C) is measured directly with a test strip at room temperature using
a reagent that takes advantage of the varying surface charge density on LDLs and non-LDLs to selectively make LDLC available
for testing.

Documents:

02210-kolnp-2006-assignment.pdf

02210-kolnp-2006-correspondence-1.1.pdf

02210-kolnp-2006-form-3-1.1.pdf

02210-kolnp-2006.claims.pdf

02210-kolnp-2006.correspondence others.pdf

02210-kolnp-2006.description(complete).pdf

02210-kolnp-2006.drawings.pdf

02210-kolnp-2006.form-1.pdf

02210-kolnp-2006.form-3.pdf

02210-kolnp-2006.form-5.pdf

02210-kolnp-2006.international publication.pdf

02210-kolnp-2006.international search report.pdf

02210-kolnp-2006.pct form.pdf

2210-KOLNP-2006-(27-08-2012)-CORRESPONDENCE.pdf

2210-KOLNP-2006-(27-08-2012)-FORM-13.pdf

2210-KOLNP-2006-(27-08-2012)-FORM-16.pdf

2210-KOLNP-2006-(27-08-2012)-OTHERS.pdf

2210-KOLNP-2006-ABSTRACT 1.1.pdf

2210-KOLNP-2006-ABSTRACT.pdf

2210-KOLNP-2006-AMANDED CLAIMS.pdf

2210-KOLNP-2006-CLAIMS 1.1.pdf

2210-KOLNP-2006-CORRESPONDENCE 1.2.PDF

2210-KOLNP-2006-DESCRIPTION (COMPLETE) 1.1.pdf

2210-KOLNP-2006-DESCRIPTION (COMPLETE).pdf

2210-KOLNP-2006-DRAWINGS 1.1.pdf

2210-KOLNP-2006-DRAWINGS.pdf

2210-KOLNP-2006-FORM 1 1.1.pdf

2210-KOLNP-2006-FORM 1.pdf

2210-KOLNP-2006-FORM 13.pdf

2210-kolnp-2006-form 18.pdf

2210-KOLNP-2006-FORM 2 1.1.pdf

2210-KOLNP-2006-FORM 2.pdf

2210-KOLNP-2006-FORM 3.pdf

2210-KOLNP-2006-FORM-27.pdf

2210-KOLNP-2006-OTHERS.pdf

2210-KOLNP-2006-PETITION UNDER RULE 137.pdf

2210-KOLNP-2006-REPLY TO EXAMINATION REPORT.pdf

abstract.02210-kolnp-2006.pdf


Patent Number 246648
Indian Patent Application Number 2210/KOLNP/2006
PG Journal Number 10/2011
Publication Date 11-Mar-2011
Grant Date 08-Mar-2011
Date of Filing 04-Aug-2006
Name of Patentee POLYMER TECHNOLOGY SYSTEMS, INC
Applicant Address 7736 ZIONSVILLE ROAD, INDIANAPOLIS, IN 46268, U.S.A.
Inventors:
# Inventor's Name Inventor's Address
1 LAWRENCE GREGORY M 5539 ELKHORN DR., APT. 115, INDIANAPOLIS, IN 46254, U.S.A.
2 PASQUA JOHN 10 CATALINA CIRCLE, ZIONSVILLE, IN 46077, U.S.A.
PCT International Classification Number C12Q 1/60,G01N 33/92
PCT International Application Number PCT/US2005/003234
PCT International Filing date 2005-02-03
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/541,681 2004-02-03 U.S.A.
2 10/962,272 2004-10-11 U.S.A.