EXHIBIT 1014
`
`US. PATENT NO. 5,215,886 TO PATEL ET AL.
`
`lnfopia Ex. 1014 pg. 1
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`,
`United States Patent
`'
`Patel et al.
`
`[19]
`
`'
`
`[54] HDL DETERMINATION IN WHOLE BLOOD
`[76]
`Inventors: P. Jivan Patel, 1235 Wildwood Ave.
`,
`.
`.
`(#102), Sunnyvale, Cale. 94089,
`.
`Mchael P. Allen, 677 W. Garland
`.
`.
`Tet" sunnyvale’ Cal1f. 94°86;
`Prithipal Singh, 25627 Elena Rd.,
`'
`Los Altos Hills, Calif. 94022
`The portion of the term of this patent
`Silbsefiluem t9 SeP- 25. 2007 has been
`disclaimed.
`-
`_
`121] APPL N03 516,528
`[22] Filed:
`Nov. 21’ 1990
`
`[ "' ] Notice:
`
`.
`
`[63]
`
`Related US. Application Data
`.
`‘
`.
`'
`~
`fiz’gm‘gt’z’mngm £215.“- No‘ 95713451 ;May 24}
`' a a" one . w c
`‘5 3 ”mm“ lon'm'pm °
`Ser. No. 353,910, May 18, 1989, Pat» No. 4,959,324,
`which is a continuation-in-part of 851. No. 195,881,
`May 19, 1988, Pat. No. 4,999,237, and a continuation.
`in-part of Ser. No. 64,883, Jun. 22, 1987, Pat. No.
`4,973,549.
`
`lllllllllllllllllllllllllllllllllllllIllllllllllllllllllllllllllllIllllllll'
`.
`U5005215886A
`.
`[11] Patent Number:
`5,215,886
`[45] Date of Patent:
`Jun. 1, 1993
`.
`
`:1:
`
`.
`
`[51]
`
`‘
`
`[56]
`
`Int. Cl.5 ......................... C12Q 1/60; C12Q 1/26;
`,
`Cle 1/28; GOIN 21/00
`[52] US. Cl. ........................................ 435/11; 422/56;
`435/25; 435/28; 436/71; 436/169; 436/170
`.
`[58] Field of Search ....................... 435/11, 19, 25, 28;
`436/71 824 169 170- 422/551
`’
`’
`’
`’
`References Cited
`U.S. PATENT DOCUMENTS
`4,959,324 9/1990 Ramel et 11.1.
`........................ 436/169
`Pr:
`Ex
`M h
`l G W h
`‘mary
`aminer— ic ae
`.*
`itys yn
`Assistant Examiner—Ralph Gitomer
`, Attorney, Agent, or Firm—Bertram I, Rowland
`[57]
`ABSTRACT
`
`High density lipoprotein-cholesterol determination is
`made'employing a device which allows for removal of
`red blood cells measurement of sample‘volume re-
`’
`.
`.
`’
`3:10:31 Of II’DL and VLD..L’ and. quantitation 0f HDL'
`0 0 we“? 0'1 a quantitatlon SmP‘
`'
`17 Claims, 2 Drawing Sheets
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`10
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`lnfopia Ex. 1014 pg. 2
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`US. Patent
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`June 1, 1993
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`Sheet 1 of 2
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`5,215,886
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`an.mm.mm
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`K
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`Infopia Ex. 1014 pg. 3
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`US. Patent
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`June 1, 1993'
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`Sheet ~2 of 2
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`5,215,886
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`F
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`Iii/1931?;
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`lnfopia Ex. 1014 pg. 4
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`1
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`5,215,886
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`- 2
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`I-IDL DETERMINATION IN WHOLE BLOOD
`
`RELEVANT LITERATURE
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation-in-part of applica-
`tion Ser. No. 357,045 filed May 24, 1990, now aban~
`doned, which is a continuation-in-part of application
`Ser. No. 353,910, filed May 18, 1989 now US Pat. No.
`4,959,324, which in turn was a continuation-in-part of
`application Ser. No. 195,881 filed May 19, 1988', now_
`US Pat. Nos. 4,999,287, and 64,883 Jun. 22, 1987, now
`US. Pat. No. 4,973,549.
`‘
`‘
`
`INTRODUCTION
`
`1. TechnicalField
`The field of this invention is noninstrumented quanti-
`tative determination of high-density lipoprotein—choles-
`terol in whole blood;
`2. Background
`High-density lipoprotein (HDL) consists of a number
`of heterogeneous particles that vary with respect to
`size, content of lipid, and apolipoprotein. The major
`apolipoproteins found in HDL are A0) and A01) and
`these proteins constitute about 90% of total HDL pro-
`tein. The roles of HDL in lipid transport are (l) to act
`as a reservoir of C apoprotein required for triglyceride
`transport; (2) to act as a “scavenger” of surplus choles-
`terol and phospholipids liberated from lipolysed tri-
`glyceride-rich lipoproteins; and (3) to transport surplus
`cholesterol from peripheral tissues to the liver for ex-
`cretion and catabolism, both directly and indirectly, via
`other lipoproteins and the lipid transfer proteins.
`HDL concentration has been feund to correlate in-
`versely with coronary heart disease. Epidemiologic
`studies have emphasized the importance of HDL as a
`negative risk factor. There is,
`therefore, substantial
`demand for an easy method for quantitation of this
`lipoprotein. Currently,
`the determination of HDL—
`cholesterol
`is extremely difficult and impractical
`to
`‘quantitate directly. The primary methods depend on the
`measurement of the plasma content of HDL.-cholesterol
`after selective separation. Several methods are available
`for separation, such as ultra-centrifugation, electropho-
`resis, precipitation as in soluble complexes between
`lipoproteins, polyanions and divalent cations,- gel or
`membrane filtration;.and precipitation with antibodies
`to the apolipoproteins.
`Ultracentrifugation, followed by precipitation with
`heparin and manganese chloride is the most commonly
`used reference method. Ultraeentrifugation separates
`VLDL on the basis of differential density, while hepa-
`rin-manganese chloride removes LDL by precipitation.
`HDL is estimated as cholesterol in the plasma fraction
`of a density greater than 1.063 g/ml. Ultra-centrifuga-
`tion requires expensive instrumentation and significant
`technical skill and has therefore found limited applica-
`tion. Electrophoretic techniques lack precision and
`' accuracy in the range of 20—40 mg/dL, where the great-
`est clinical interest lies. Gelpermeation chromatogra-
`phy is too complex and time consuming for routine
`analysis. There is, therefore, a clear interest in the de-
`velopment of techniques having relatively simple proto-
`cols and equipment for the quantitative determination
`of cholesterol in high-density lipoprotein.
`
`10
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`15
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`30
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`35
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`65.
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`See also Allen, M. P., Delizza, A., Raine], U., Jeong,
`H., and Singh, P. A Non-instrumented Quantitative Teri
`System and its Application for Determining Cholesterol
`Concentration in Whole Blood, Clin. Chem. 1990; 36(9)
`1591—1597; and 'Rarnel, U., Allen, M. P., 'and Singh, P.
`Sample Pad Assay Initiation Device and Method ofMak-
`ing U.S. Pat. No. 4,959,324, issued Sep. 25, 1990.
`SUMMARY OF THE INVENTION
`
`is quantitated
`High-density lipoprotein-cholesterol
`from blood by removal of apoB containing lipoproteins
`with a membrane, followed by enzymatic reaction of
`cholesterol and cholesterol esters to produce hydrogen
`peroxide. The hydrogen peroxide is then quantitated on
`a quantitation strip to which a~coupling dye is bound,
`which reacts with the product of the peroxidase to
`produce a detectable colored product.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIGS. la and lb are diagrammatic side views of a
`separation device and the quantitation strips, respec-
`tively; and
`FIG. 2 is a diagrammatic plan view of a base plate
`and slide of an alternative quantitation device. .
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`Method and devices are provided for the determina-
`tion of high-density lipoprotein-cholesterol
`(HDL-
`cholesterol) from blood. The methodand device pro-
`vide for initial removal of a substantial proportion of the
`red blood cells present in the blood, removal of substan-
`tially all of the very low density lipoprotein (VLDL)
`and «low density lipoprotein (LDL), which lipoproteins
`contain apo B lipoprotein. The resulting HDL-choles~
`terol concentrated plasma fraction is then treated with
`enzymes which react with cholesterol and cholesterol
`esters to produce hydrogen peroxide. Preferably, the
`hydrogen peroxide is then measured by transport of the
`hydrogen peroxide on a quantitation strip in conjunc-
`tion with a peroxidase, where a compound is conju-
`gated to the strip. The reaction of the peroxidase with a
`coupling compound results in the coupling compound
`reacting with another compound bound to the strip
`with production of a highly colored product. The dis-
`tance of the colored front from the sample site or other
`designated position is quantitatively related to the
`amount of HDL-cholesterol in the blood sample.
`. A series of membranes are provided which allow for
`a number of functions in producing the desired sample,
`which is free of red blood cells and free of cholesterol
`associated with lipoproteins other than HDL. The first
`stage is the application of the blood sample to one or
`more membranes, desirably having diminishing pore
`size. The membranes are selected to efficiently remove
`red blood cells without lysis. Significant lysis of. red
`blood cells results in the discoloration of the sample,
`which discoloration interferes with the measurement
`for the determination of 'HDL—cholesterol and more
`importantly hemoglobin may chemically interfere with
`the assay system. Generally, the pore size will be in the
`range of 0.5 to 50 um, whereas with two filters, the first
`filter will have a pore size in the range of about 5 to 50
`um, while the second filter will have a pore size in the
`range of about 0.5 to 10 pm. The filters are selected to
`minimize the sample holdup in the filter and should
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`lnfopia Ex. 1014 pg. 5
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`5,215,886
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`3
`substantially reduce the red blood cells (RBC) which
`are transferred to the next membrane layer.
`The next membrane layer nonnally serves to remove
`VLDL and LDL, which'include apolipoproteins B, C
`and E and various techniques may be employed for the
`removal of the cholesterol associated with VLDL and
`LDL. Alternatively one may use two treated glass fibre
`membranes for removal of LDL and VLDL at the same
`time as the RBC are being removed.
`Another‘ possibility is the use of either one or more
`filters (filter 1 and/or 2) to remove VLDL and LDL,
`while at the same time separating the red blood cells
`(see FIG. 1a).
`~
`In a preferred embodiment, a membrane comprising a.
`divalent cation containing anionic polymer, which min-
`imizes red blood cell lysis and efficiently separates the
`VLDL and LDL from the HDL, is employed. Illustra-
`tive of such polyanion products are dextran sulfate
`having magnesium as the counterion, or sodium heparin
`having divalent manganese as the counterion. Of partic-
`ular interest, therefore, are polymers of at least about
`5,000 molecular weight, usually at least about 50,000
`molecular weight, particularly heparin (5—20 kDal) or
`dextran (25—500 kDal) which are sulfuric acid deriva-
`tives, sulfonates or sulfates, in conjunction with divalent
`cations, particularly magnesium and manganese. The
`polymers may be covalently or non-covalently bound
`to a porous substrate, so long as the polymers are re-
`tained by the porous substrate during the subject proce-
`dure. Various substrates may be employed, such as glass
`fiber, paper, synthetic membranes, or the like. The vari-
`ous substrates may be activated for covalent bonding by
`a variety of conventional agents, such as silane, e.g.,
`aminopropyltriethoxysilane, with glass fiber, activated,
`e.g., carbodiimide-activated, paper or synthetic mem-
`branes, or the like. The amount of polymer bound to the
`membrane may be readily determined by determining
`the efficiency with which the HDL is separated from
`the VLDL and LDL and the loss of HDL on the mem-
`brane. The polymers may be bound to any or all of the
`porous filtration substrates. That is in a preferred em-
`bodiment, the VLDL and LDL precipitating reagent
`may be bound to polymeric membrane, filter 1, filter 2,
`membrane 3 or it may be bound to the sample receiving
`pad. The precipitating reagents may be bound onto any
`combinations of 2 or 3 or all porous substrates. (See
`FIG. 1)
`Usually, the amount of polymer will be about 0.001 g
`to 0.5 g per cmz. The membrane will generally have a
`thickness in the range of about 10 um to 1000 pm and a
`pore size in the range of about 0.1 to 50 um.
`‘
`Instead of using precipitating reagents, affinity rea-
`gents may be employed, such as monoclonal or poly- ‘
`clonal antibodies specific for apolipoproteins B, C, and
`E. The use of the antibodies also allows the determina~
`tion of LDL uncontaminated with HDL, by employing
`antibodies specific for the apo A protein on the LDL,
`which will allow passage of the HDL through the mem-
`brane.
`,
`The antibodies may be bound to the membranes de-
`scribed above by analogous techniques, using carbodi-
`imide, cyanogen bromide, diazo compounds, activated
`olefins for reaction with thiol' groups, or the like. The
`amount of antibodies bound to the membrane will vary
`with the affinity of the antibody, the number of active
`sites, the molecular weight of the antibody, e.g., IgM or
`IgG, or the like. Usually, the amount of antibody will be
`in the range of about 0.1 ug to 100 pg per cmz. These
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`antibodies may be immobilized on the polymeric mem-
`brane, filters 1 or 2 or membrane 3 or on the sample
`receiving pad (FIG. 1). The polymeric membrane may,
`be positioned above, or in between filters 1 and 2, or
`membrane 3.
`There may be one or more additional membranes to
`separate the sample receiving pad from the LDL re-
`moving membrane, to control flow, to further remove
`particulate matter, or for other purposes. These mem-
`branes will not be reactive, generally having pore sizes
`in the range of about 0.5 pm to 1.0,p.m.
`Illustrative membranes which may find use for re-
`moval of red blood cells include 8&8 Glass 30, What-
`man GFD, S&S 3662, Pall glass fiber membranes, Sar-
`torius cellulose acetate, Filterite polysulfone asymmet—
`ric membrane, Ultrabind 450, Nucleopore, etc. Mem-
`branes which find use for removal of LDL are illus-
`trated by Whatman GFD, Whatman 31ET or Ultrabind
`450.
`.
`The sample is received by a sample pad. The sample
`pad may serve a number of functions. The sample pad,
`in conjunction with other components, may serve to
`measure the volume of the sample. The sample pad will
`usually have a volume ofabout 1 to 60 ill and a thick-
`ness of about 0.1 to 5 mm, In addition, the sample pad
`may have one or more reactants, particularly enzyme
`reactants bound to the pad. The sample pad will initially
`be protected from contact with the quantitation strip.
`Various mechanisms may be employed to provide a
`barrier for contact between the sample pad and the
`quantitation strip, which barrier may be moved to allow
`for contact between the sample pad and the quantitation
`strip.
`7
`~
`The sample pad may conveniently be a bibulous
`membrane, which will absorb the plasma sample and
`serve as a bridge for transport of eluent from an eluent
`source through the sample pad to conversion pad and
`subsequently to the quantitation strip and allow for
`substantially quantitative transport of cholesterol (in-
`cluding cholesterol precursors) or an enzymatic reac-
`tion product of cholesterol from the reaction pad to the
`quantitation strip.
`While it is preferred that one or more of the mem-
`branes serve to remove VLDL and LDL, the reagent
`may be present solely on the sample pad, solely on the
`membrane for removing RBCs, combinations thereof,
`or on all or some of the components of the device in.
`which the plasma contacts before undergoing substan-
`tial enzymatic reaction. Where the enzymes are on the
`pad, the reagent for removing the VLDL and LDL will
`not be restricted to the sample pad.
`The cholesterol is measured by reaction with a com-
`bination of enzymes, cholesterol esterase and choles-
`terol oxidase. The es'terase provides for hydrolysis of
`cholesterol esters to cholesterol,'while cholesterol oxi-
`dase provides for oxidation of cholesterol with oxygen
`to hydrogen peroxide. The enzyme may be present on
`the pad, may be upstream from the pad in a reaction
`zone prior to the quantitation region of the quantitation
`strip, or a'combination thereof. The enzymes may but
`are not required to be non-diffusively bound to the
`surface, so that the enzymes will substantially remain at
`the site where they are positioned prior to the beginning
`of the assay.
`‘
`The sample pad may be protected from acting as a
`bridge and contacting the quantitation strip in a variety
`of ways. In one embodiment, a removable plastic bar-
`rier may be inserted between the sample pad, the quanti-
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`~
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`Infopia Ex. 1014 pg. 6
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`5
`tation strip, and the strip providing the source of eluent.
`The plastic barrier may include a mesh strip, e.g., Ni-
`tex ®, 100—50011, for wiping the pad of excess sample.
`In another embodiment, the pad may be at a site distant
`from the quantitation strip and the eluent providing
`strip, where after receiving the sample, the sample pad
`is moved into contact with the two strips. In both of
`these cases, various wiping means may be employed to
`remove excess sample from the pad, so that a substan—
`tially reproducible amount of sample will be absbrbed
`by the pad.
`The eluent-supplying strip may be of any convenient
`bibulous material which can be dipped into an eluent
`and wick the eluent up to the sample pad, when the '
`15
`sample pad is in contact with the eluent source strip
`Various papers may find use, such as cellulose strips,
`e.g.., chromatography paper, silica on a support, alu-
`mina on a support, polymeric membranes, such as nitro-
`cellulose and nylon Generally, the eluent source strip
`will be not less than about 0.5 cm and not more than
`about 25 cm
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`5
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`10
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`20
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`The quantitation strip may serve, as already indi-
`cated, in providing a reaction zone, where the choles-
`terol esters and enzymes are present which will react
`with the cholesterol to produce the reactant, hydrogen
`peroxide Thus, a zone may be provided where the
`cholesterol will react to produce hydrogen peroxide,
`which will then be transported by the eluent to the
`‘ quantitation region. The quantitation region is charac-
`terized by having one member of a dye couple bound to
`the region at a Concentration which allows for a reason-
`able dynamic range associated with the concentration
`range of interest of HDL. For the most part, the con-
`centration range of' interest is from about 20 to 100
`mg/dL of HDL-cholesterol, so that one wishes to have
`at least about 10 mm, per 25 mg/dL change in conCen-
`tration, preferably. at least about 7 mm per 25 mg/dL
`and not more than about 20 mm,,usually not more than
`about 15 mm per 25 mg/dL.
`'
`‘
`The quantitation strip will generally have dimensions
`of about 5 mm to 130 mm and may be of the same or
`different material from the eluent source strip. Passively
`adsorbed to the strip will be one member of a dye cou-
`ple, such as MBTH or AAP. The coupling member may
`be any of a variety of substrates for horseradish peroxi-
`dase, which can couple with the other member bound to
`the strip. Illustrative coupling members include N,N-
`dimethylaniline; 5-(N-methylanilino pentanoyl ethylene
`diamine; N-ethylaniline; N-ethylmeta-toluidine; 2-(N-
`ethyl-meta-toluidino)ethanol; N-ethyl-N-(Z-hydroxy-S-
`sulfopropyl)-meta-toluidine sodium salt; N-ethyl, N—sul-
`fopropyl-meta-toluidine
`sodium salt; 2-(Nethyl-m;
`toluidino)ethanol, N-ethyl meta-toluidine; 1,8-dihy-
`droxy-3,6-dimethoxynaphthalene' or analogs thereof.
`These compounds are characterized by being capable of
`reacting in the presence of a peroxidase with hydrogen
`peroxide to form a compound which may be coupled
`with MBTH to form an indamine dye, which allows for
`detection of a color front in relation to the amount of
`hydrogen peroxide produced from the cholesterol in
`the sample.
`The eluent will comprise the horseradish peroxidase,
`the cdupling member, usually buffers, andgother miscel-
`laneous additives as appropriate, e.g., PVP, non-inter-
`fering proteins, detergents, particularly‘nonionic deter-
`gents, antifungal agents, etc. The concentration of the
`horseradish peroxidase will generally be in the range of
`about 0.2911/1111 to 1.611/ml, usually about 2—14 rig/ml.
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`5,215,886
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`Buffers will generally provide a pH in the range of
`about 4 to 9, with a buffer concentration that will be
`sufficient to provide the necessary‘buffering, generally»
`being in the range of about 10 to 500 mM. Illustrative
`buffers include phosphate, Tris, MOPSO, MOPS, bo-
`rate, carbonate, etc.
`In carrying out the assay, a blood sample may be
`placed on the topmost membrane and gravity and capil-
`lary action provide the transport force. The blood will
`migrate through the red blood cell separating mem-
`brane(s), so that the sample is substantially free of red
`blood cells when it contacts the membrane for removal
`of cholesterol present as other than HDL. The plasma
`sample will pass through the reactant membrane, so as
`to remove LDL and VLDL and leave HDL as the only
`protein-bound cholesterol. In a second embodiment the
`red blood cell separating membranes and the treated or
`reactant membranes, which removes VLDL and LDL,
`are the same. The sample receiving pad may also be
`used as a reactant membrane, The sample will then
`migrate to the sample pad and be absorbed by the sam-
`ple pad. When the sample pad is at least saturated, the
`excess fluid will be removed as described above by
`moving the pad or other instrumentality, which serves
`to wipe the pad of the excess fluid. The pad will now be
`incontact with the eluent source strip and the quantita-
`tion strip The eluent will move through the eluent
`source strip, using the pad as a bridge and carry choles-
`terol, cholesterol esters, and/or enzymatic products of
`cholesterol up to be quantitation region, where the
`hydrogen peroxide will react with the coupling agent to
`activate the coupling agent
`to react with the other
`member of the dye bound to the Quantitation strip in the
`quantitation region. The extent of the color produced
`from an arbitrary point at the beginning of or prior to
`the quantitation region may be related to the amount of
`HDL—cholesterol1n the sample. By using known stan-
`dards, the distance of the color front from some origin,
`can be directly related to the amount of HDL-choles-
`terol1n the samplein a quantitative manner.
`Turning now to FIGS. la and 1b and 2, the subject
`invention will be further described. The separation
`device 10 of FIG. 1:: has a well~12 in which is situated
`in the direction of flow, polymeric membrane 14, filter
`1, 16, filter 2,18 membrane 3, 20 and polycarbonate
`membrane 22 The separation and well sit over sample
`pad 24
`The polymeric membrane 14 serves to remove red
`blood cells without lysis, while filters 1 and 2, 16 and 18,
`respectively, are activated to remove VLDL and LDL,
`while allowing I-IDL to pass through To further1m-
`prove filtering, membrane 20 is provided to further
`ensure removal of VLDL and LDL, where membrane
`3 may be activated1n the same or different way. from
`filters 1 and 2.
`A polycarbonate membrane 22 serves to 'prevent
`contact between membrane 3, 20 and sample pad 24.
`As depicted in FIG. 2 sample pad 24 is moved from a
`first position as shown1n the FIG. 2 to a second posi-
`tion, as shownin FIG. 1b, where the sample pad 24
`comes into contact with the conversion area 28 and
`wicking strip 26 to act as bridge to allow flow of fluid
`from the Wieldng strip 26 through the sample pad 24,
`conversion area 28, to the quantitation strip 30
`The sample on sample pad 24 is carried by wicking
`buffer from wicking strip 26 into conversion area 28'in
`which enzymes are presentto convert cholesterol and
`cholesterol esters to hydrogen peroxide. The Wicking
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`lnfopia Ex. 1014 pg. 7
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`7
`buffer then carries the hydrogen peroxide into the quan-
`titation area 30, where the hydrogen peroxide reacts
`with dyes under catalysis with by horseradish peroxi-
`dase. The extent of color formation is related to the
`amount of HDL present as indicated by the amount of 5
`cholesterol present in the HDL.
`The measurement device may be fabricated from
`three injection molded parts or by any other convenient
`process. The parts comprise a base plate 40, a slide 42
`and a clear cover plate, not shown. The base plate 40
`consists of a cutout to accept the slide 42, a slot 46 with
`locating pins 48 into which the conversion area 28,
`quantitation strip 30 and wicking strip 26 are precisely
`positioned, maintaining about a 2 mm gap 44 between
`them, and a well 50 designed to capture the released
`transport solution, e.g., wicking buffer.
`The slide 42 consists of a vented receptor site 52 into
`which the reagent 24 pad is inserted and over which the
`separation device 10 is placed. An arm 54 with dual
`shearing designed to facilitate the release of the trans-
`port solution from a pouch which is housed in a well of
`the cover plate, and asnap 56 to lock the slide in place,
`once pulled are provided. The cover plate consists of a
`well 32, which houses a sealed foil pouch (not shown)
`containing the transport solution. The cover plate has
`an orifice for placement of the separation device 10 for
`the introduction of the sample. The cover plate also
`comprises a squeegee metering bar which serves to
`control the volume of sample absorbed by the sample
`pad 24.
`The following examples are offered by way of illus-
`tration and not by way of limitation.
`EXPERIMENTAL
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`10
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`Experimental Details
`Standard Curve
`
`- 1. An assay is performed by using mylar laminated
`chromatography strips containing the quantitation re-
`gion (70X 5 mm), conversion region (7X 5 mm), sample
`pad (7 x5 mm) and wicking strip (12X5 mm). The
`wicking reagent is (0.1M) MOPSO buffered (pH 7.0)
`' protein solution containing HRP (5 pg/ml). The paper
`employed for the quantitation region is 0.1 mg/ml
`MBTH paper and human serum samples equivalent to
`25, 50, 75 and 100 mg/dL of HDL were employed.
`The assay is carried out as follows:
`(a) 0.01 ml of plasma or serum is deposited on the
`sample pad; (b) wicking is initiated by allowing the
`lower portion of the assay strip to contact 0.5 ml
`wicking reagent contained in a test tube. The assay
`is complete when the wicking solution reaches the
`end of the measurement region. The strips are then
`removed and the height to which the colored band
`is formed is plotted against cholesterol concentra-
`tion. A substantially linear plot was obtained, with
`the migration height varying from about 15 mm to
`about 40 mm.
`
`35
`
`45
`
`50
`
`55
`
`The assay was also performed with hydrogen perox-
`ide calibrators equivalent to 25, 50, 75 and mo mg/dL.
`The plot of migration height versus hydrogen peroxide
`incubation was substantially linear with migration
`height varying from about 22 mm to about 48 mm
`2. Gel Electrophoresis
`AccuMeter ® cassettes A (FIG. 2) (see U.S. applica-
`tion Ser. No. 353,910 filed May 18, 1989, now U.S. Pat.
`No. 4,959,) with treated and untreated membranes are
`used for this study. Filters 1, 2, membrane 3 and/or a
`
`65
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`5,215,886
`
`8
`combination of 1, 2, 3 or all may be treated with the
`precipitating reagents.
`The assay involves the application of whole blood or
`its plasma (0.04 ml) to the sample site on the cassette.
`Following a two minute incubation the sample pad is
`removed from the cassette and its plasma contents
`squeezed out (0.005 ml) and applied to an Agarose gel.
`After the last sample has been applied to the gel, 10
`mins. are allowed for diffusion. The gel is placed into a
`Paragon electrophoresis cell and electrophoresed for 50
`ruins. at 100 volts. Upon completiOn of electrophoresis,
`the gel is removed from the Paragon electrophoresis
`cell and placed into a fixative solution for 5 mins.
`The gel is removed frbm the'fixative solution and
`dried until completely dry. The dried gel is processed in
`the following sequence:
`——-——____.___________
`Lipoprotein working stain
`5 minutes
`Dwain Solution I
`3 dips
`Desmin Solution 11
`3 dips
`Denim Solution III
`5 minutes
`——-—_____.__.___—_
`
`The gel is finally rinsed in a deionized water and then
`completely dried. Eight samples were electrophoresedr
`1 whole blood (cassette with untreated membranes; 2
`and 3 whole blood (cassette with treatedmembranes); 4
`plasma (cassette with untreated membranes); 5 and 6
`plasma (cassette with treated membranes); 7 and 8 su-
`pernatant of plasma following LDL and VLDL precip-
`itation using phosphotungstate/Mg2+. With the excep-
`tion of sample 1 and 4, using untreated cassettes which
`showed the presence of pre-B and [3 subunits, all of the
`samples only showed the presence of the a subunit.
`
`Cholesterol Analyses
`
`Cholesterol concentrations are determined following
`in situ precipitation in AccuMeter ® cassettes (FIG. 2).
`AccuMeter cassettes with treated and untreated mem-
`branes are used for this study. Filters 1, 2, membrane 3
`and/or a combination of 1, 2, 3 or all may be treated
`with the precipitating reagents. Boehringer Mannheim
`Diagnostic high performance reagent (BMDHP) is used
`to measure the plasma cholesterol collected on the sam- .
`pie pad. BMD Preciset aqueous based cholesterol cali-
`brators equivalent to 50, 100, 150, 200, and 300 mg/dL
`are used for this study.
`Whatman glass fibers GF/D(6.0 um and/or What-
`man 31ET chromatography paper were used to prepare
`treated membranes. The precipitating reagent solutions
`were prepared as follows: (1) Into deionized water (50
`ml) was dissolved 0.5 g dextran sulfate (Na salt, Mw 500
`kD), followed by the slow addition of 4.26 g anhydrous
`Mng with stirring, (2) Into 30 ml deionized water was
`dissolved 0.421 g heparin (Na salt, 176 USP units/mg)
`and 2.97 g MnClz.
`,
`The precipitating reagent (12 ml) was poured into a
`lasagne dish tilted at 45°. The membrane material (10
`cmX7 cm)’was slowly and uniformly passed through
`the reagent solution. (The GFD membrane was sup-
`ported by Nitex nylon screen of the same dimension.)
`The membrane was then dried in an oven at 80° C. for
`about 15 min being inverted occasionally for unifor-
`mity. Discs (5 mm dia.) were then cut.
`For solution phase precipitation, the dextran sulfate
`solution was prepared as described above, the heparin
`solution was twice as concentrated, and the phos-'
`photungstate/Mg2+ solution was prepared by diluting _
`
`I
`
`lnfopia Ex. 1014 pg. 8 ‘
`
`
`
`
`
`

`

`
`
`$215,886
`
`10
`departing from the spirit or scope of the appended
`claims.
`
`9
`4:1 BMD-HDL-cholesterol reagent (0.55M phospho-
`tungstic acid; 25 mM MgC12) in deionized‘water.
`The assay involves the application or plasma or serum
`(0.04 ml) to the sample site on the cassette. Following a
`two minute incubation, the slide is pulled and the sam-
`pie receiving pad removed and placed in the BMDHP
`reagent (1 ml). The plasma or serum sample on the pad
`(0.005 ml) isextracted for one hour at room tempera-
`ture. The-solution is then measured at A500 nm. A
`standard curve is generated by pipetting BMD choles-
`terol calibrators (0.005 ml) into the BMDHP reagent (1
`ml), incubating for 15 mins. and then measuring the
`absorbance at 500 nm.
`
`5
`
`10'
`
`LDL and VLDL precipitation was also performed in
`test tubes using the precipitating reagents and assay 15
`protocol listed below:
`—-———g————-——_—_______
`Ppt Reagent
`.
`' Ppt Reagent Vol.
`Sample Vol.
`Phosphotungstate/
`0.5 ml
`0.2 ml
`M3011 (diluted 4:1
`in H20)
`Dextran SOMMgClz
`0.05 ml
`‘
`0.5 ml
`Heparin/MnClz
`0.05 ml
`0.5 ml
`
`
`20
`
`What is claimed is:
`l. A method of determining the amount of high den-
`sity lipoprotein-cholesterol (“HDL-cholesterol”) in a
`blood sample, said method comprising:
`passing a blood sample through a first membrane to
`remove substantially all red blood cells without
`significant lysis of said red blood cells to provide
`plasma free of red blood cells and red blood cell
`components at an assay determination interfering
`level;
`passing said plasma through a membrane system com—
`prising at least one porous filtration membrane;
`collecting and measuring said plasma on a sample
`Pad;
`bringing said sample pad in fluid transferring relation~
`ship with an eluent source strip and a quantitation
`strip, wherein at least one of said sample pad and a
`region of said quantitation strip proximal to said
`sample pad comprises non-diffusibly bound choles-
`terol esterase and cholesterol oxidase, wherein said
`quantitation strip comprises a dye forming com-
`pound capable oi‘ reacting with an oxidized cou-
`' pling compound to produce an intensely colored
`dye evenly distributed along said quantitation strip
`downstream from said cholesterol esterase and
`cholesterol oxidase;
`eluting said sample and any enzymatic reaction prod-
`uct with an eluent comprising a peroxidase and said
`coupling compound, by contacting said eluent
`source strip with said eluent;
`wherein a reagent for selectively removing very low
`density and low density lipoprotein is bound to at
`least one of said first membrane, a membrane of
`said membrane system or said sample pad, while
`_ allowing said HDL-chlolesterol
`to substantially
`quantitatively pass through said second membrane
`whereby any cholesterol in said blood is oxidized to _
`hydrogen peroxide and said coupling compound is
`enzymatically oxidized to couple with said dye
`_ forming compound to form a colored region along
`said quantitation strip of a length proportional to
`the amount of HDL-cholesterol in said blood.
`‘
`2. A method according to claim 1, wherein said mem»
`brane system comprises dextran sulfate or heparin with
`a divalent cation salt wherein said cation is magnesium
`or manganese.
`.
`y
`,
`3. A method according to claim 2, wherein said diva-
`lent cation salt is manganese chloride.
`‘
`4. A method according to claim 2, wherein said diva-
`lent cation salt is manganese chloride together with
`heparin.
`'
`‘ 5. A method according to claim 1, wherein said mem-
`brane system comprises antibodies to very