WO2004025265A2 - Bande d'essai et procede permettant de determiner la concentration du cholesterol ldl a partir d'un echantillon de sang total - Google Patents

Bande d'essai et procede permettant de determiner la concentration du cholesterol ldl a partir d'un echantillon de sang total Download PDF

Info

Publication number
WO2004025265A2
WO2004025265A2 PCT/US2003/029102 US0329102W WO2004025265A2 WO 2004025265 A2 WO2004025265 A2 WO 2004025265A2 US 0329102 W US0329102 W US 0329102W WO 2004025265 A2 WO2004025265 A2 WO 2004025265A2
Authority
WO
WIPO (PCT)
Prior art keywords
ldl
cholesterol
ldl cholesterol
sample
test strip
Prior art date
Application number
PCT/US2003/029102
Other languages
English (en)
Other versions
WO2004025265A3 (fr
Inventor
Bruce Shull
Hyeon-Sook Lee Zeng
Sunil Anaokar
Gena Lynn Antonopoulos
Original Assignee
Polymer Technology Systems, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Polymer Technology Systems, Inc. filed Critical Polymer Technology Systems, Inc.
Priority to CA002499058A priority Critical patent/CA2499058A1/fr
Priority to MXPA05002955A priority patent/MXPA05002955A/es
Priority to EP03754634A priority patent/EP1540353A4/fr
Priority to AU2003272453A priority patent/AU2003272453A1/en
Publication of WO2004025265A2 publication Critical patent/WO2004025265A2/fr
Publication of WO2004025265A3 publication Critical patent/WO2004025265A3/fr

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/525Multi-layer analytical elements
    • G01N33/526Multi-layer analytical elements the element being adapted for a specific analyte
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/04Endocrine or metabolic disorders
    • G01N2800/044Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • the present invention relates generally to testing of body fluids for concentration of analytes and more particularly to test strips for determining concentration of analytes in whole blood.
  • Total cholesterol includes low density lipoproteins (LDL), very low density lipoproteins (NLDL) and high density lipoproteins (HDL). It is well- established from epidemiological and clinical studies that there is a positive correlation between levels of LDL cholesterol ("bad” cholesterol) and coronary heart disease and a negative correlation between levels of HDL cholesterol ("good” cholesterol) and coronary heart disease. Standing alone, the level of total cholesterol in blood, which is a measure of the sum total of HDL, LDL, NLDL and chylomicrons, is not generally regarded as an adequate indicator of the risk of coronary heart disease because the overall level of total cholesterol does not reveal the relative proportions of HDL, LDL and NLDL. To better assess the risk of heart disease, it is desirable to determine the amount of LDL cholesterol in addition to total cholesterol.
  • LDL low density lipoproteins
  • NLDL very low density lipoproteins
  • HDL high density lipoproteins
  • 1718-0004 calculation involves measurements other than LDL cholesterol, it is subject to potential compounded inaccuracies from the determinations of the other lipids in the equation. Further, its usefulness is limited when assaying blood samples with triglycerides levels above 400 mg/dl. Ultracentrifugation is a known technique to separate LDL cholesterol, but it is tedious, time consuming, and the highly labile lipoproteins can be substantially altered by the high salt concentrations and centrifugal forces. "Furthermore, a plethora of different types of equipment and tubes are used, making conditions difficult to reproduce from one laboratory to another and consistent separations highly dependent on the skills and care of the technician.” Id. at 238.
  • the '827 patent discloses a two-stage liquid phase reaction to quantify LDL concentration in a fluid sample.
  • the sample containing LDL cholesterol is placed in a first reagent which includes trimethyl ⁇ -cyclodextrin as a sugar compound, polyoxyethylene monolaurate as a protein solubilizing agent,
  • EMSE N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamene
  • Tris buffer Tris buffer
  • the sample is then 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 cholesterol is then calculated by separately subjecting a standard solution of cholesterol to the same procedure and comparing the respective absorbance values.
  • This method suffers from the drawback of requiring conducting the reaction at a temperature of 37 °C. Further, this method requires individual reagents to be added in two distinct steps at two different times
  • cholesterol esterase and cholesterol oxidase act on the test sample in the presence of a surfactant which acts on lipoproteins other than LDL ("non-LDLs").
  • a surfactant which acts on lipoproteins other than LDL ("non-LDLs").
  • the hydrogen peroxide thereby generated is decomposed to water and oxygen by catalase.
  • a phenol-based or an aniline-based hydrogen donor compound is reacted with the hydrogen peroxide to convert it to a colorless quinone.
  • Preferred surfactants which act on the non-LDLs include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether, polyoxyethylene octyl phenyl ether, polyoxyethylene nonylphenyl ether, and the like.
  • cholesterol remaining in the test sample which should theoretically contain only LDL, is quantified.
  • the second step may be carried out by adding a surfactant which acts on at least LDL and quantifying the hydrogen peroxide by the action of the cholesterol esterase and the cholesterol oxidase added in the first step.
  • one disadvantage of the method taught by the '164 patent is that it requires conducting the reaction at a temperature of 37 °C, and it has been found that the test is not accurate at lower temperatures.
  • Another disadvantage of the '164 patent similar to the '827 patent is that the '164 patent requires individual reagents to be added in two distinct steps at two different times.
  • a more general disadvantage of these liquid phase LDL tests is that they are not easily adaptable to point of care (“POC”), much less over the counter (“OTC”) applications.
  • the present invention provides a dry phase test strip and method for determining the concentration of LDL in whole blood or plasma.
  • the test strip directly measures concentration of total cholesterol and directly measures concentration of non-LDLs, the difference therebetween being equal to the concentration of LDL cholesterol.
  • Dry phase test strips of the present invention function at room temperature and all test results are produced from pseudo endpoint reflectance measurements, such that the test method does not require timing.
  • the present invention provides a method of determining concentration of non-LDL cholesterol in a whole blood sample using a dry phase test strip.
  • the whole blood sample is contacted with a blood separation layer of the test strip and the blood cells are separated from the sample, thereby producing plasma.
  • the plasma so produced is then contacted with a test layer and the non- LDL fraction reacts substantially faster than the LDL fraction to produce color substantially in proportion to the concentration of LDL cholesterol in the sample.
  • the color produced is measured and corresponds to the concentration of non-LDL cholesterol.
  • the test layer employs a surfactant which acts on lipoproteins other than LDL, "non-LDLs,” such that the non-LDLs react to produce color and the result is read photometrically before any LDLs have substantially reacted.
  • non-LDLs lipoproteins other than LDL
  • the reflectance or colorimetric response is a function of non-LDL concentration, substantially unaffected by LDL concentration.
  • the present invention provides a test strip for determining the concentration of LDL cholesterol in a sample of whole blood or plasma.
  • the test strip includes a test strip matrix having at least two stacks. A first one of the stacks has reagents incorporated therein to produce a colorimetric response in proportion to the amount of total cholesterol in the sample. A second one of the stacks has reagents incorporated therein to produce a color response in proportion to the amount of non-LDL cholesterol in the sample. The value of non- LDL cholesterol obtained from the second stack can be subtracted from the value of total cholesterol obtained from the first stack to yield the value of LDL cholesterol in the sample.
  • One advantage of the present invention is that it provides a dry phase test strip that produces reliable measurements of LDL cholesterol without relying on the Friedewald equation. As noted above, the Friedewald equation has serious drawbacks.
  • Another advantage of the present invention is that the inventive test strips can be used over a range of room temperatures, quite unlike the known homogeneous liquid LDL assays, which require heating to 37°C. This temperature independence of the present invention is a significant advantage because test strips required to be heated to a specified temperature would be largely unmarketable in the over the counter (“OTC”) and point of care (“POC”) markets and, of course, inconvenient.
  • OTC over the counter
  • POC point of care
  • Still another advantage of the present invention is that the non-LDL test is completed and the result read in less than 1 Vz minutes.
  • test strips in accordance with the present invention that read multiple analytes, e.g., HDL, total cholesterol and non-LDL, the three results are all obtained at about the same time. Waiting for the non-LDL result is unnecessary.
  • the present single measurement approach is in contrast to the prior art LDL assays noted above that teach two-stage reactions and a result measured after both stages.
  • Still another advantage of the present invention is that it provides a dry phase "lipid panel" which measures total cholesterol, HDL cholesterol and LDL
  • 1718-0004 cholesterol results without reliance upon the Friedewald equation, and without the need to measure triglycerides, which was hitherto not possible in a dry phase test strip.
  • the present invention provides a dry-phase test strip for determining LDL concentration in whole blood or plasma that is inideally quicker, more convenient, and is essentially time and temperature independent. This breakthrough technology makes possible for the first time the potential for point-of- care and patient self-testing of LDL cholesterol without relying upon the Friedewald equation.
  • Fig. 1 is a perspective view looking down on an assembled and closed test strip in accordance with the present invention
  • Fig. 2 is an exploded perspective view of a test strip holder in accordance with the present invention, the view being taken from the bottom of the test strip holder;
  • Fig. 3 is perspective view of a test strip holder in accordance with the present invention, the test strip holder having its top and bottom portions unfolded and the underside thereof being shown;
  • Fig. 4 is an exploded perspective view of a test strip holder in accordance with the present invention illustrating the layers and stacks of the test matrix and their relationship with the top and bottom portions of the test strip holder;
  • Fig. 5 is a side sectional view of an exemplary test matrix in accordance with one embodiment of the present invention.
  • Fig. 6 is a graph illustrating color production versus time for the two stage reaction that occurs in the non-LDL stack of panel of test strips in accordance with the present invention
  • Fig. 7 is a perspective view illustrating the vertical flow scheme utilized by the stacks and blood separation layer of the present invention.
  • Figs. 8, 9 and 10 are cross-sectional views of the layers of test strips used in certain of the examples given hereinbelow.
  • Figs. 11 and 12 are cross-sectional views of the layers of test strips in accordance with alternate embodiments of the present invention.
  • HDL high density lipoprotein.
  • LDL low density lipoprotein.
  • VLDL very low density lipoprotein.
  • NonHDL refers to LDL, NLDL and chylomicrons, Le ⁇ , lipoproteins other than HDL that will react with a conventional cholesterol reaction membrane.
  • ⁇ on-LDL refers to HDL, NLDL and chylomicrons, he., lipoproteins other than LDL that will react with a conventional cholesterol reaction membrane.
  • Plasma refers to the non-cellular portion of blood from which cellular components such as red blood cells are excluded.
  • “Serum” technically differs from plasma, in that it does not include fibrinogen. However, for purposes of this application “serum” and “plasma” are sometimes used interchangeably. "Room Temperature” means from about 17°C to about 30°C.
  • test strip 20 includes test strip holder 22 which is preferably formed by injection molding.
  • Test strip holder 22 includes handle 24 and top portion 26 (Figs. 2 and 3) which is preferably hingedly attached by hinge portion 28 to bottom portion 30, shown exploded away in Fig. 2.
  • top portion 26 is foldable about hinge portion 28 over bottom portion 30 as shown.
  • Top portion 26 includes an opening 32 while bottom portion 30 includes three spaced openings 34.
  • Opening 32 while shown as round, can be formed as an elongated oval shape to facilitate disbursement of blood.
  • opening 32 in holder 22 defines an area for depositing a body fluid sample while openings 34 define areas in which optoelectronic measurements
  • openings 34 can be configured with transparent windows, although such is not necessary.
  • test strip described herein is suitable for use with a modified optoelectronic instruments sold under the trademarks BioScanner and Cardio Chek, available from Polymer Technology Systems, Inc., Indianapolis, IN.
  • Test matrix 36 is made up of a top disbursement layer 38, a blood separation layer 40, stacks 42, and adhesive layer 44 having openings 46 that align with openings 34 and the bottoms of respective stacks 42 when the layers are assembled.
  • Stacks 42 are further made up of one or more vertically aligned layers, the function and specifics of which are described in further detail hereinbelow.
  • the second layer of the "stacks" 42 in Fig. 4 is shown in phantom to indicate that this second layer is not used in all embodiments disclosed in this application.
  • Opening 32 exposes a part of disbursement layer 38 and openings 34 and 46 expose the bottom surface of the bottom layers of stacks 42.
  • portions 26 and 30 have complementary I-shaped indentations or recesses 48 (Figs. 2 and 3) in which the corresponding I-shaped matrix 36 is received. Recesses 48 allow portions 26 and 30 to be snapped together in a snap-tight engagement as shown in Fig. 1 while still exerting a minimally compressive force on matrix 36.
  • top portion 26 includes oval shaped receptacles 50 that receive complementary shaped bosses 52 disposed on portion 30.
  • Receptacles 50 include pegs 54 that fit via friction fit into mating cylindrical openings 56 formed in bosses 52.
  • Stacks 42 all include the same number of layers or at least have about the same thickness, such that the bottom surfaces of stacks 42 are substantially coplanar. This coplanar structure helps maintain the proper compressive pressure on matrix 36 by holder 22.
  • the amount of pressure with which matrix 36 is to be pressed together is a design variable that can be adjusted by (1) adjusting the depth of recesses 48; (2) adjusting the amount of pressure with which matrix 36 is to be pressed together
  • the top layer of matrix 36 is a disbursement or spreader layer capable of rapidly spreading the blood sample 58 rapidly through layer 38 as shown by the reference arrows. It has been found that layers used as conjugate pads in pregnancy test kits perform quite well as layer 38.
  • Layer 38 is an open cell layer capable of rapidly and effectively spreading the fluid sample.
  • One suitable material for layer 38 is available under the name "Accuflow Plus-P" from Schleicher & Schuell, Inc.
  • Another suitable material for layer 38 is available under the name "Accuwik” from Pall Biochemicals. Both of these layers are made of polyester and provide excellent movement of blood sample 58 through layer 38.
  • layer 40 is a blood separation layer that is adjacent to and in fluid communication with layer 38. Blood separation layer 40 separates blood cells from plasma and passes the plasma therethrough, retaining the blood cells. Blood separation layer 40 is generally a glass fiber membrane.
  • a suitable commercial membrane for layer 40 is Ahlstrom Grade 144, thickness 0.378mm, available from Ahlstrom Filtration, Inc., Mt. Holly Springs, PA. Other glass fiber matrices could be substituted as demonstrated in the examples that follow hereinbelow. More specifics regarding the blood separation membrane are given in co-pending U.S. provisional patent applications serial nos. 60/344,300 and
  • the stack 92 is formed of a single layer and is spaced from stack 94 and is adjacent to and in fluid communication with layer 40. Stack 92 takes plasma from layer 40 and produces a color response proportional to the concentration of total cholesterol in sample 58.
  • stack 96 is also a "total cholesterol" stack, identical to stack 92. The preparation of reagents for the total cholesterol stacks (also called “panels") is set forth in detail in the examples hereinbelow.
  • middle stack or layer or panel 94 forms a color response that is proportional to the amount of non-LDL cholesterol in sample 58, at room temperature and without requiring the reaction to be timed, as will be explained below.
  • Layer 94 is loaded with reagents such that non-LDL cholesterol produces a color response much faster than does LDL cholesterol.
  • the preparation of reagents for the non-LDL stack or panels is set forth in detail in the examples hereinbelow.
  • the non-LDL cholesterol layer 94 differs from the cholesterol layer primarily in that layer 94 includes a surfactant which acts on non-LDLs, i.e., lipoproteins other than LDL.
  • a surfactant which acts on non-LDLs, i.e., lipoproteins other than LDL.
  • a commercially available and suitable surfactant is sold under the trade name Emulgen B66.
  • Emulgen B66 is sold under the trade name Emulgen B66.
  • the reaction that produces color from non-LDL cholesterol is significantly faster than the reaction that produces color from LDL cholesterol, particularly at the lower room temperatures used by the present invention.
  • an optoelectric instrument which uses a pseudo end-point algorithm, as disclosed in U.S. patent no. 5,597,532 effectively detects such endpoint after the non-LDLs in the plasma provided to layer 94 have reacted, but before the LDL cholesterol has significantly contributed to color production. That is, an end-point can be detected while the reaction producing color from LDL cholesterol is ongoing.
  • the "pseudo end-point" is reached when there is only a small change in color density per time interval, e.g., five (5) seconds.
  • the algorithm can be programmed to reach a pseudo endpoint when change in reflectance drops to less than 1% over 5 seconds.
  • This pseudo endpoint chemistry allows measuring the reflectance and thus non-LDL concentration without timing the reaction, which is a significant advantage.
  • layer 94 becomes wetted with plasma and the non-LDLs in the sample begin to produce color quickly as shown by the curve in Fig. 6.
  • time t ls ⁇ seudo endpoint shown on the curve) the nonLDLs have substantially completely reacted to form color, but the LDL cholesterols have not. Nonetheless, the reaction of the LDLs after ti is much slower.
  • the algorithm detects an endpoint at the time the slope flattens as shown at ti.
  • the difference in reaction rates of non-LDLs versus LDLs produces a "pseudo endpoint" sufficient to form a cut-off point for the algorithm, which is a significant and surprising advantage. It is significant in that there is no need to establish a predetermined time which corresponds to the completion of non-LDL color production. It is surprising because the liquid phase tests, from which the dry phase tests were adapted, required strict control of the time at which the non-LDL measurement was taken, which is consistent with the homogeneous prior art assays described above that require the first phase to be timed.
  • the pseudo endpoint is enhanced by conducting the test at lower temperatures, viz., room temperature, in stark contrast to the prior art teachings of heating the liquid reagents to 37°C. Lower temperatures are believed to inhibit the slow phase (LDL color production) sufficiently such that the slope of the LDL production curve shown in Fig. 6 is sufficiently flat. Yet, at the same time, the first phase of the
  • reaction in which the non-LDLs are expended to produce color, is nonetheless sufficiently fast and ends sufficiently abruptly such that the pseudo endpoint shown in Fig. 6 is always detected at room temperature.
  • test strip in accordance with the present invention were used at elevated temperatures, e ⁇ g., 37°C, as taught by prior art liquid phase tests, the second stage LDL reaction would take place more quickly, as reaction kinetics are typically enhanced by higher temperatures.
  • a faster second phase is desirable in the liquid phase tests discussed above because it shortens the total test time, which even at 37°C can be quite long.
  • the pseudo endpoint is not as prominent and can therefore be missed by the algorithm. See dashed line corresponding to elevated temperatures in Fig. 6.
  • the elevated temperatures taught by prior art liquid phase LDL assays teach away from the present invention.
  • the present inventive dry phase test strips never measure LDL concentration directly.
  • the length of time required to complete the second phase of the reaction, in which LDLs react to produce color is not important, whether it be 2 minutes or 20 minutes.
  • this novel test strip does not require the LDL concentration to be directly measured, only a single step of the two phase reaction occurring in test layer 94 is measured, thereby completely eliminating one of the two sequential measurements taught and indeed required by certain of the prior art homogeneous assays discussed above.
  • pH of the impregnating solution of layer 94 should be pH 7. Selectivity for non-LDLs decreases as the pH becomes lower than 7.
  • Example 1 illustrates the adaptation of the test from the liquid phase and the reliance on pH.
  • Reagent la 100 mM Citric Acid, pH 5 0.5% Triton X-100
  • Reagent lb 100 mM Citric Acid, pH 5 0.5% Emulgen B66* 0.56 mM MAOS
  • Reagent 1 100 mM MOPS Buffer, pH 7.0 0.5% Emulgen B66* 0.56 mM MAOS
  • Emulgen B66 is a nonionic surfactant available from Kao Corporation (1-3, Bunk 2-Chome; Sumida-Ku; Tokyo 131-8501, Japan). Emulgen B66 has previously been shown to have a selective action for the reactivity of Non-LDL Cholesterol (cited in US; US 6,194,164). Triton X100 is used as a nonselective surfactant to give a Total Cholesterol Reaction.
  • Total cholesterol was measured with Reagent la (pH 5/Triton X100) and non-LDL cholesterol was measured separately using either Reagent lb (pH 5 Emulgen B66) or lc (pH 7.0/Emulgen B66). Measurements were made at various pre-selected times after initiating the reaction with Reagent 2. The optimum Reaction time was determined to be 75 seconds. LDL Cholesterol was calculated as the difference between Total and Emulgen B66 reactive Cholesterol.
  • LDL Cholesterol was measured using a commercially available Kit: LDL Direct Liquid Select Cholesterol Reagent. This kit was run according to the manufacturer's directions using a Cobas Mira clinical analyzer. This reaction is performed in two steps . In the first step sample is mixed with a reagent that solubihzes only non-LDL Cholesterol. During this step, non-LDL is removed with a reaction that does not produce color. Then a second reagent is added to produce color with the remaining LDL Cholesterol.
  • Emulgen B66 gives selectivity for non-LDL at pH 7 but does not give selectivity at pH 5.
  • Agreement between Reference and Measured LDL decreased if other reaction time intervals were used. It was found that at 75 seconds, substantially all of the non-LDL Cholesterol had reacted but little or no LDL Cholesterol had reacted. At longer measurement intervals, the measured values of LDL Cholesterol decreased due to the slow but significant reaction of LDL Cholesterol in the
  • Formulations for impregnation of reaction membranes were made according to the Tables below.
  • a Foundation Solution containing a portion of the ingredients was made and used in both the Total and Non-LDL formulations.
  • Test strips were assembled using test strip holders as described in co-pending provisional patent application serial no. 60/342,790. The layers in the strip holders from top to bottom are shown in Figs. 8 and 9 and are spreading mesh 200 (Tetko); blood separation layer 202; Cytosep 1660 (untreated blank) 204 and Biodyne A membrane impregnated with the either Total Cholesterol (206) or non-LDL Cholesterol formulation (208).
  • Strip holders with two reaction zones were used to assemble test strips with both Total and Non-LDL Cholesterol reaction pads.
  • a cross section of the layers is shown in Fig. 10.
  • the layers from top to bottom were a spreading layer 300 consisting of Accuflow PS (Schleicher&Schuell), blood separation layer 302 and reaction membranes 304 and 306 composed of Biodyne A impregnated with either Total Cholesterol Reagent (304) or Non-LDL Cholesterol Reagent (306).
  • the spreading layer 300 spread the sample evenly and delivered it vertically to the blood separation layer 302, which in turn retained red blood cells before delivering plasma to reaction layers 304 and 306.
  • LDL cholesterol was calculated with the test strips by subtracting the measured non-LDL from the measured total cholesterol.
  • Strip holders with three reaction zones were used to assemble test strips with both total and non-LDL Cholesterol reaction pads or stacks as shown in Fig. 5.
  • the layers from top to bottom were a spreading layer 38 made from Accuflow PS
  • Non-LDL cholesterol reaction membrane 94 was positioned as the center panel or stack and the two outer panels 92 and 96 were impregnated with the total cholesterol solution. Results were computed with by averaging the two total cholesterol obtained from photometrically reading the color production from layers 92 and 96 or by using a single total cholesterol value from layer 92 or 96. In either case, only a single non- LDL value was obtained from the color production in layer 94.
  • Matrix 36' shown in Fig. 11 includes three stacks that are spaced apart and are adjacent to and in fluid communication with disbursement layer 38. Each stack has its own blood separation layer 440 as its top layer. The difference between matrix 36' and matrix 36 (Fig. 5) is that matrix 36' has separate blood separation
  • matrix 36' is the same as matrix 36 described with reference to Fig. 5.
  • the embodiment shown in Fig. 11 is advantageous in that layers 440 collectively consume less blood than all of layer 40 (Fig. 5), which may help reduce the quantity of blood required to complete the test.
  • Experimental data show that either the separate blood separation layers as shown in Fig. 11 or a single blood cell separation layer as shown in Fig. 5 produce accurate results.
  • Fig. 12 illustrates a panel including 4 stacks.
  • One embodiment for this panel could include a total cholesterol stack, an HDL stack, a non-LDL stack and a triglycerides stack.
  • the top layer is a spreader layer 536, as described above with reference to Fig. 5, except slightly longer so as to accommodate four stack.
  • the first (total cholesterol), third (non-LDL) and fourth (triglycerides) stacks include a spacer layer 502, as taught in co-pending provisional application serial no. 60/344,300.
  • the spacer layers can be formed from CytoSep 1660 and serve to keep the stacks even, since layer 512 is needed in the HDL stack to precipitate and separate non- HDLs, as also taught in co-pending application serial number 60/344,300.
  • the HDL and triglycerides stacks are fully disclosed and taught in co-pending application serial number 60/344,300, as is the total cholesterol stack.
  • the non-LDL stack is only disclosed in this application.
  • the four-stack system just described can incorporate an error checking system by incorporating the Friedewald equation described above.
  • an opto-electronic instrument such as Cardio-Chek, available from Polymer Technology Systems, Inc., can be programmed to calculate the values of total cholesterol, LDL, HDL and triglycerides in a sample being tested by strip 536. Then, using the Friedewald equation, the instrument can calculate the value of LDL and compare it to the measured value, the measured value being the measure total cholesterol value less the measured non-LDL. If the two values differ by a predetermined percentage, an error signal can be produced. For example, the display on the instrument can instruct the user to repeat the test.
  • the value of NLDL cholesterol plus chylomicrons can be determined by taking the measured non-LDL value and subtracting therefrom the measured value of HDL.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Endocrinology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention a trait à une bande d'essai en phase sèche (20) et à un procédé permettant de déterminer la concentration du cholestérol LDL dans un échantillon de sang total ou de plasma. La bande d'essai (20) selon l'invention comprend un empilement (92) ou une feuille, qui mesure la concentration du cholestérol total, et un autre empilement (94) ou feuille, qui mesure la concentration du cholestérol « non LDL », soit la somme du cholestérol HDL, du cholestérol VLDL, et des chylomicrons. La différence entre les valeurs obtenues correspond à la concentration de cholestérol LDL. Les bandes d'essai en phase sèche (20) selon l'invention fonctionnent à température ambiante, et tous les résultats de l'essai sont produits à partir de mesures du facteur de réflexion effectuées lors d'un pseudo-point de virage, si bien qu'il n'est pas nécessaire de chronométrer le procédé d'essai. L'invention se rapporte également à la production d'une feuille à lipides améliorée, qui indique la concentration, dans un échantillon sanguin, du cholestérol HDL, du cholestérol total et du cholestérol LDL, et ce sans faire appel à la formule de Friedewald.
PCT/US2003/029102 2002-09-16 2003-09-16 Bande d'essai et procede permettant de determiner la concentration du cholesterol ldl a partir d'un echantillon de sang total WO2004025265A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002499058A CA2499058A1 (fr) 2002-09-16 2003-09-16 Bande d'essai et procede permettant de determiner la concentration du cholesterol ldl a partir d'un echantillon de sang total
MXPA05002955A MXPA05002955A (es) 2002-09-16 2003-09-16 Tiras de prueba y metodo para determinar la concentracion de colesterol de lipoproteinas de baja densidad a partir de sangre completa.
EP03754634A EP1540353A4 (fr) 2002-09-16 2003-09-16 Bande d'essai et procede permettant de determiner la concentration du cholesterol ldl a partir d'un echantillon de sang total
AU2003272453A AU2003272453A1 (en) 2002-09-16 2003-09-16 Test strip and method for determining ldl cholesterol concentration from whole blood

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US41120902P 2002-09-16 2002-09-16
US60/411,209 2002-09-16

Publications (2)

Publication Number Publication Date
WO2004025265A2 true WO2004025265A2 (fr) 2004-03-25
WO2004025265A3 WO2004025265A3 (fr) 2004-07-01

Family

ID=31994248

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/029102 WO2004025265A2 (fr) 2002-09-16 2003-09-16 Bande d'essai et procede permettant de determiner la concentration du cholesterol ldl a partir d'un echantillon de sang total

Country Status (6)

Country Link
EP (1) EP1540353A4 (fr)
AU (1) AU2003272453A1 (fr)
CA (1) CA2499058A1 (fr)
MX (1) MXPA05002955A (fr)
WO (1) WO2004025265A2 (fr)
ZA (1) ZA200502214B (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008086019A1 (fr) * 2007-01-09 2008-07-17 Cholestech Corporation Dispositif et procédé de mesure du cholestérol associé au ldl
US7582484B2 (en) 2002-01-18 2009-09-01 Cholestech Corporation High-density lipoprotein assay device and method
US7772007B2 (en) 2004-04-02 2010-08-10 Cholestech Corporation Assay device for direct measurement of LDL cholesterol
US7795038B2 (en) 2002-04-09 2010-09-14 Cholestech Corporation High-density lipoprotein assay device and method
JPWO2009031506A1 (ja) * 2007-09-05 2010-12-16 アークレイ株式会社 低密度リポタンパク質(ldl)コレステロール測定方法およびldlコレステロール測定用試験片

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5132086A (en) * 1990-02-06 1992-07-21 Chemtrak Corporation Non-instrumented cholesterol assay
US5135716A (en) * 1989-07-12 1992-08-04 Kingston Diagnostics, L.P. Direct measurement of HDL cholesterol via dry chemistry strips
US5460974A (en) * 1992-10-13 1995-10-24 Miles Inc. Method of assaying whole blood for HDL cholesterol

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3193634B2 (ja) * 1996-05-29 2001-07-30 第一化学薬品株式会社 Ldlコレステロールの定量方法
JP3441993B2 (ja) * 1999-01-27 2003-09-02 松下電器産業株式会社 コレステロールセンサ
US6524864B2 (en) * 2000-12-28 2003-02-25 Aurora L. Fernandez Decastro Test strip for simultaneous detection of a plurality of analytes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5135716A (en) * 1989-07-12 1992-08-04 Kingston Diagnostics, L.P. Direct measurement of HDL cholesterol via dry chemistry strips
US5132086A (en) * 1990-02-06 1992-07-21 Chemtrak Corporation Non-instrumented cholesterol assay
US5460974A (en) * 1992-10-13 1995-10-24 Miles Inc. Method of assaying whole blood for HDL cholesterol

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1540353A2 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7582484B2 (en) 2002-01-18 2009-09-01 Cholestech Corporation High-density lipoprotein assay device and method
US7795038B2 (en) 2002-04-09 2010-09-14 Cholestech Corporation High-density lipoprotein assay device and method
US7772007B2 (en) 2004-04-02 2010-08-10 Cholestech Corporation Assay device for direct measurement of LDL cholesterol
WO2008086019A1 (fr) * 2007-01-09 2008-07-17 Cholestech Corporation Dispositif et procédé de mesure du cholestérol associé au ldl
US7824879B2 (en) 2007-01-09 2010-11-02 Cholestech Corporation Device and method for measuring LDL-associated cholesterol
JPWO2009031506A1 (ja) * 2007-09-05 2010-12-16 アークレイ株式会社 低密度リポタンパク質(ldl)コレステロール測定方法およびldlコレステロール測定用試験片
US8304204B2 (en) 2007-09-05 2012-11-06 Arkray, Inc. Method for measuring low-density lipoprotein (LDL) cholesterol

Also Published As

Publication number Publication date
AU2003272453A1 (en) 2004-04-30
WO2004025265A3 (fr) 2004-07-01
ZA200502214B (en) 2006-05-31
EP1540353A4 (fr) 2007-01-24
MXPA05002955A (es) 2005-10-18
EP1540353A2 (fr) 2005-06-15
CA2499058A1 (fr) 2004-03-25

Similar Documents

Publication Publication Date Title
US20040126830A1 (en) Test strip and method for determining LDL cholesterol concentration from whole blood
EP0799896B1 (fr) Bandes de test pour la détermination de glucose dans le sang
EP1474692B1 (fr) Batonnet diagnostique permettant de determiner une concentration de plusieurs analytes dans un echantillon de fluide unique
EP0800082B1 (fr) Bandes de test pour la détermination de glucose dans le sang
EP0769558B1 (fr) Bandelette d'essai pour la détermination de glucose dans le sang à sensibilité réduite pour l'hématocrite
JP7096456B2 (ja) 血液サンプルのアッセイ法
EP0852336A1 (fr) Bandes de test à lecture visuelle
US7824879B2 (en) Device and method for measuring LDL-associated cholesterol
MXPA97002503A (en) Reagent test stress to determine glucose in the san
MXPA97002502A (en) Reagent test for the determination of glucose in the san
EP1792189B1 (fr) Dispositif d'essai servant a determiner la concentration en cholesterol ldl dans un echantillon
ZA200502214B (en) Test strip and method for determining LDL cholesterol concentration from whole blood
EP0526226B1 (fr) Procédé et dispositif pour exécuter des essais biologiques contenant un système pour tamiser des particules

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2499058

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 200502214

Country of ref document: ZA

Ref document number: 2003754634

Country of ref document: EP

Ref document number: 2005/02214

Country of ref document: ZA

Ref document number: PA/a/2005/002955

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 2003272453

Country of ref document: AU

WWP Wipo information: published in national office

Ref document number: 2003754634

Country of ref document: EP

NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP