CA1056282A - Multilayer analytical elements for use in the assay of cholesterol - Google Patents

Abstract

Abstract of the Disclosure An analytical element for use in the quantitative assay of complex fluids for cholesterol content comprising a support, a reagent layer comprising a substance having cholesterol oxi-dase activity dispersed in a suitable binder coated on one side of the support, and a porous medium comprised of one or more layers which serve to spread the sample in a manner providing a substantially constant volume of sample per unit area to the reagent layer in spite of variation in the volume of sample applied to the element coated over the reagent layer. According to a preferred embodiment, the reagent layer also contains an indicator system which reacts with one or more products of the cholesterol oxidase induced decomposition of cholesterol to produce a spectrophotometrically quantizable energy absorption shift. According to a further preferred embodiment, the indicator system comprises a substance having peroxidative activity and a composition which undergoes an energy absorption shift, pre-ferably a color change, in the presence of hydrogen peroxide and said substance having peroxidative activity. Another preferred embodiment provides for the inclusion of a cholesterol ester hydrolyzing system comprising a lipase having cholesterol esterase activity and a protease in either the porous medium or the reagent layer to hydrolyze cholesterol esters which may be present in the complex solution under analysis.
In a highly preferred embodiment, the enzymatic cho-lesterol ester hydrolyzing system and the cholesterol oxidase are contained in the porous medium which is separated from the reagent layer containing the indicator system by a hydrophilic barrier layer.

Description

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Field of the Invention ~ he present invention relates to an improved element for the quantitative analysis of cholesterol in complex aqueous solutions, particularly blood serum.
Background of the Invention As is well known to those skilled in the art, the ; quantitative analysis of cholesterol in complex mixtures such as blood serum which contains both free and esterified cholesterol, has generally involved the handling of corrosive chemicals to hydrolyze the cholesterol esters to free cholesterol and generally complex and not easily automated techniques for analyzing for free cholesterol once hydrolysis has been completed and the cholesterol isolated.
, Belgian Patent No. 801,742, issued January 2, 1974, describes a multilayer element for use in the quantitative analysis of complex fluids such as blood serum and urine, which element comprises a support, a reagent layer which contains a test reagent which undergoes a quantifying reaction with a component of the fluid being analyzed coated over the support, and, on the opposite side of the reagent layer from the support a porous medium comprised of one or more layers through which the component of the sample undergoing reaction is transmitted to the reagent ~ i layer; the porous medium being adapted to spread the sample in a manner providing a substantially constant volume of sample per unit area to the reagent layer.
U. S. Patent No. 3,869,349 of Goodhue and Risley describes a totally enzymatic method for the hydrolysis of `
cholesterol esters using a lipase having cholesterol esterase `
activity and a protease.

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, U. S. Patent No. 3,884,764 in the names of Goodhue, '~ Risley, and Snoke describes a totally enzymatic quantitative ;
;~ single solution assay for cholesterol in complex solutions containing both free and esterified cholesterol using the fore-~-`. going cholesterol hydrolysis technique of Goodhue et al combined t~ , with a cholesterol oxidase oxidation of free cholesterol prepared either as described in U. S. Patent No. 3,909,359 of Goodhue and Risley, or German Offenlegungsschrift 2,246,695 published March 26, 1973. The solution of U. S. Patent No. 3,884,764 may also include a hydrogen peroxide detection system based on the action of peroxidase on a color indicator system.
German Offenlegungsschrift No. 2,246,695 published March 26, 1973, describes the use of a cholesterol oxidase enzyme different from that described in the aforementioned Goodhue and Risley U. S. Patent No. 3,909,359 to assay for free cholesterol.
The method of this German publication is a solution method and still requires the handling of corrosive materials to hydrolyze the cholesterol esters which may be present in blood serum in addition to the sometimes unwieldy handling of solution to obtain the assay.
Summary of the Invention The present invention provides an element for use in the quantitative analysis of cholesterol in complex solutions.
The use of this element requires substantially no handling of corrosive chemicals and requires only a minimum of operator participation to obtain highly accurate, reproducible results.

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According to the present invention, there is provided an analytical element for use in the quantitative assay of complex fluids for cholesterol content comprising a support, a reagent layer comprising a substance having cholesterol oxidase activity dispersed in a suitable binder, and a porous medium, comprised of one or more layers which serve to spread the sample in a manner providing a substantially constant volume of sample per unit area to the reagent layer in spite of variation in the volume of sample applied to the element. According to a preferred embodiment the reagent layer also contains an indicator system which reacts with one or more products of the cholesterol oxidase induced decomposition of cholesterol to produce a spectrophoto-metrically quantizable energy absorption shift, preferably a color change. According to a further preferred embodiment, the indicator system comprises a substance having peroxidative activity and a composition which undergoes an energy absorption shift, preferably a color change, in the presence of hydrogen peroxide and the substance having peroxidative activity. Another pre-ferred embodiment provides for the inclusion of a cholesterol ester hydrolyzing system comprising a lipase having cholesterol esterase activity and a protease in either the porous medium or the reagent layer to hydrolyze cholesterol esters which may be present in the complex solution under analysis.
In a highly preferred embodiment, the enzymatic choles-terol ester hydrolyzing system and the cholesterol oxidase are contained in the porous medium which is separated from the reagent layer containing the indicator system by a hydrophilic, ., .
~ hydrogen peroxide permeable barrier layer.
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Description of the_Drawings Figures 1 - 5 depict alternative configurations of pre-... .
ferred embodiments of the analytical web of the present invention.

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Figure 6 shows graphic results of analyses performed with the webs of the present invention.
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Detailed Description of the Invention The basic structure of the elements of the present in-vention is described in Belgian Patent No. 801,742, issued January 2, 1974.
As described therein, the multilayer analytical element is designed primarily for use in automated quantitative analysis and is simple in structure, easily manufactured at reasonable cost, and adapted to carrying out analyses in continuous analyzers in a simple and effective manner. This analytical element may be utilized in the form of a long continuous strip or tape and, for convenience in describing the present invention, is generally re-ferred to herein as an analytical tape. However, it will be apparent that the element can be used in other forms, such as sheets or short strips or in the form of small sections or chips , . .
which may or may not be mounted in aperture cards or other hold-ing devices, and all such forms of the element are intended to be within the scope of the present invention. As hereinafter described, the multilayer element incorporates within a discrete reagent layer at least some of the test reagents needed for carrying out the analysis so that in use the operator merely needs to provide for proper application of the sample which is to be analyzed. Automated dispensers for applying a controlled amount of sample to the element at the appropriate location are known and any such dispenser may be utilized with the element of this invention. Quantitative analysis for cholesterol in, for example, blood serum, is readily accomplished by use of con-ventional spectrophotometers or other quantizing techniques capable of measuring in a rapid manner and with high degrees of accuracy the amounts of reaction products present, as will be described more fully hereinafter.

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The analytical tape is an integral multilayer element which provides all the layers needed for carrying out the various functions involved in the analytical process. It is comprised of a support, a reagent layer which contains one or more test re-`~ agents which will be described in detail below, and a porous medium, comprised of one or more layers, for performing the function of spreading. According to a preferred embodiment of the invention, the porous medium also serves the purposes of filtering the sample and facilitating reflective spectrophoto-metric analysis through the support side of the tape. Since the element is an integral multilayer structure in which the support ~i and the various layers are bonded together, and since in use it is only necessary to apply the sample to be analyzed to the top layer of the element and direct the element through an appropri-ate analyzer, preferably a spectrophotometer of one type or another, the equipment utilized in the automated analysis pro-cedure need not be complex and the inherent simplicity of the ` system of analysis is fully realized.
The support used in the multilayer analytical element of ` 20 the present invention is comprised of a radiation (preferably uv or visible light) transmitting, liquid impermeable material. As .. .. .
long as it meets these criteria, the particular material used as the support is not important. A variety of polymeric materials are well suited for this purpose, such as, for example, cellulose ' acetate, poly(ethylene terephthalate), polycarbonates, or poly- -styrene. The support may be of any suitable thickness typically from about 2 to about 10 mils. As will be described more fully below, it is desirable that the support be capable of trans-mitting at least certain wavelength bands of electromagnetic .~ 30 radiation in the range of between 200 and 900 nm. According to specifically preferred embodiments, it may be desirable to have -:

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the support selectively permeable to specific relatively narrow wavelengths and opaque to all other wavelengths.
The reagent layer may be coated directly on the support or a subbing layer having energy transmission characteristics similar to those of the support may be used to aid in bonding the reagent layer to the support. The composition of the reagent layer is described in detail hereinafter. Briefly, however, it contains at least part of the test reagents intended to undergo color-forming or other radiation detectable reactions with con-stituents of the cholesterol containing sample. In this way, reaction product concentrations per unit area can be quickly and readily determined. A coating of a dispersion of one or more of the test reagents described below in a hydrophilic colloid which serves as a binder, such as gelatin, poly(vinyl alcohol), agarose, etc., is suitable as the reagent layer. Furthermore, ~;
the reagent layer may be divided into one or more discrete func-,. . .
tional layers, each of which performs a specific operation in :
the analytical procedure. For example, three discrete layers which can but need not be separated by non-interfering spacer or , 20 interlayers, could be used. The first of these could contain the reagents necessary for cholesterol ester hydrolysis, the second the "free" cholesterol assay reagents, and the third the color producing or other indicator system.

~`According to a highly preferred embodiment described below, the enzymatic hydrolysis system and the cholesterol oxidase enzyme are contained in the porous spreading layer which is in turn separated from the indicator system containing reagent layer by a hydrophilic, H2O2 permeable polymeric material. The make-up of these various layers will be described in detail below.

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On the side of the reagent layer opposite from the support are located one or more layers which perform the function of spreading the sample to distribute it uniformly in the lateral direction. According to a preferred embodiment this layer or layers, as the case may be, also serve(s) to perform the functions of (1) filtering the sample to remove components that would interfere with the occurrence or measurement of the color-forming or otherwise detectable reaction, and t2) reflecting the light transmitted through the support when quantification of the reaction product is carried out using reflective spectrophoto-metric analysis. Thus, the multilayer analytical element of the ` present invention will comprise at least two layers in addition ` to the support, one of these being the reagent layer and the other being a layer which is capable of performing the spreading function and preferably all of the aforesaid functions. However, ~ a separate layer can be used for each of the functions, if .~ .
desired, and in this instance the tape or element would comprise four layers in addition to the support. Alternatively, a single layer can be used to perform two of the three functions and a different layer to perform the third. Also, since more than one layer can be used for a given purpose, for example, two or more contiguous layers may be utilized as reagent layers, the multi- -layer tape can also be of an even more complex construction than the aforesaid four layer embodiment. Furthermore, the porous spreading layer may also be used to include a portion of the reagent. If this is desirable, certain of the reagent materials may be incorporated into this layer. Specifically, the enzymatic `~
cholesterol hydrolysis system of Goodhue et al referred to above and described in some detail below, can be incorporated into this layer to obtain cholesterol ester hydrolysis before the . : , :
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~- sample reaches the reagent layer containing the materials which act upon the total amount of free cholesterol. Furthermore, the cholesterol oxidase may also be incorporated into this layer.
The sample spreading layer in the multilayer analytical tape is in the position outermost from the support and is the ` layer upon which the liquid sample to be analyzed, such as a sample of blood serum, urine, etc., is deposited.
~; With the multilayer analytical tape described herein, .~. ..
;~;', variation in the volume of sample applied to the tape affects the diameter of the indicator spot formed in the reagent layer, 'i but the volume of liquid per unit area which reaches the reagent layer is substantially constant regardless of variations in the ~::
volume of sample applied. Accordingly, the density of the product formed in the reagent layer by the indicator reaction is ~ not significantly affected by variations in the size of the drop ,; applied to the element and is dependent only on the concentration of the component undergoing the reaction. This makes it un-~'~ necessary to apply drops of exactly uniform size to the element "~
or to know the size of the drop applied in order to obtain the desired quantitative analysis. However, careful control of drop '~ size and administration to the element are parameters, which, if ~, carefully controlled can help to enhance the accuracy of the entire system.
The function of the filtering layer which may or may not ` be present, depending upon the test being performed and the quantization mode, is to remove from the sample components that are present which would interfere with the indicating reaction in the reagent layer or would hinder quantization. Thus, in the use of the multilayer analytical element for analysis of cholesterol in whole blood, the filtering layer serves to remove _g_ ':

; red blood cells while transmitting the serum to the layer below.
In the analysis of blood serum or other fluids, the filtering layer may serve to remove unwanted components which could hinder or confuse the primary indicating reaction. The filtering layer can be comprised of any material that will provide a proper degree of porosity for the sample being analyzed, with the : optimum porosity depending upon the particular use for which the multilayer element is intended. If the element is to be used for analysis of whole blood, it is desirable that the filtering j 10 layer have a pore size of 0.5 to 5 microns.
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According to a preferred embodiment of the present inven-tion, the aforementioned spreading and filtering layer is ad-vantageously prepared by simultaneously coating two layers of a binder such as cellulose acetate dissolved in a mixed organic solvent to provide "blush" polymer layers as described below.
Such a technique simplifies the manufacturing operation by re-ducing the multiple coating of multiple layers to a single multiple coating operation while providing a highly useful spreading and/or filtering material. Optionally, if desired~
either or both of the discrete layers may contain dispersed therein a reflective pigment such as TiO2.
The physical structure of layers prepared in this fashion consists of a relatively porous upper layer which functions pri-marily as a sampling or spreading layer to provide a substan-tially constant volume of fluid sample per unit area to an under-lying layer in spite of variations in volume of sample applied (as described above), and a less porous underlayer which functions primarily as a filter layer. The porosity of these two layers is ~ -controlled during manufacture by the use of different ratios of mixed organic solvents as described in British Patent No.
134,228 or hereinafter in the discussion of "blush" polymer , . ~ : -:

; ` 1056Z82 layers. Such solvents are chosen to provide relatively low boiling, good solvents, and higher boiling poor or non solvents ~, for the specific binder used. A higher percentage of poor or non-solvents in the coating results in a coated layer of increased porosity. A particularly useful combination of sol-- vents when cellulose acetate is used as the binder comprises .
acetone, xylene, and dichloroethane in ratios of from about .
; 3.5:2:1.1 to 4.5:1:0.
. . .
~ Equipment and techniques suitable for simultaneous coating ri, lO of various individual layers within either the spreading layer or the reagent layer as described hereinabove are described in U.S. Patent No. 2,932,855 issued April l9, 1960.

According to a further preferred embodiment, the multi-.,:
layer analytical element of the present invention is adapted for ` use in an analytical system employing reflection techniques of spectrophotometric analysis, and consequently includes a layer ; which functions as a reflecting layer and thereby provides a suitable background for spectrophotometric measurement through the support side of the tape. The reflecting layer must be a 20 porous layer to permit the passage of cholesterol and cholesterol esters into the reagent layer, and should be white in order to provide an effective background for reflection spectrophotometry.
In a multilayer tape intended for analysis of whole blood, the - blood cells are blocked by the filtering layer, and yet they do not interfere with the spectrophotometric measurements, since these are made through the support with the reflecting layer serving as a suitable white background.
As hereinbefore described, a single layer can be provided which will serve the functions of sample spreading and filtering, 30 and will also serve as a reflecting layer. An example of a -suitable layer which will perform all of these functions is a ~ ?

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"blush polymer" layer. As is well known, a "blush polymer" layer can be formed on a substrate by dissolving a polymer ln a mixture ` of two liquids, one of which is a good solvent for the polymer and the other of which is of higher boiling point and is a non-solvent or at least a poor solvent for the polymer coating the .: .
` polymer solution on the substrate, and drying the coating. Since the good solvent will evaporate more readily because of its lower ; boiling point, the coating becomes enriched in the liquid which is a poor solvent or non-solvent as evaporation proceeds and, in consequence, the polymer precipitates out in the form of fine particles and forms on the substrate an adherent porous layer.
Many different polymers can be used for preparing "blush polymer"
layers for use in this invention, typical examples being poly-- carbonates, polyamides, and cellulose esters.
As an alternative to coating a "blush polymer" layer as described above, a useful layer adapted to perform the functions of sample spreading and filtering, and to provide the necessary reflective background for utilizing the analytical tape in re-flective spectrophotometry, can be provided by laminating to the reagent layer a thin layer of a microporous filter membrane.
These filter membranes are "blushed polymer" materials, made, for example, from cellulose esters, and contain pores of micro-` scopic size with a variety of materials of differing pore size being available commercially. Examples of materials of this type which are suitable for use in the present invention and which are commercially available, are the filters sold under the trade-mark Millipore by the Millipore Corporation and those sold under the trademark Metricel by the Gelman Instrument Company.
When a single layer is used to serve as a sample spreading layer, a filtering layer, and a reflecting layer, the analytical element can be comprised of only two layers and a support since ' -:
~056Z~2 the only addltional essential layer is the reagent layer. How-. .
ever, other layers could also be included, if desired. For example, two or more contiguous reagent layers, one of which con-tains the constituents necessary for hydrolysis and the other for free cholesterol assay, can be provided. Moreover, as herein-:- before described, it is within the scope of the present invention to use one layer which serves as a filtering and reflecting layer, .
and a separate layer to serve as a sample spreading layer, or to use three separate layers to carry out the functions of sample spreading, sample filtering, and light reflection, respectively.
The layers would, of course, be arranged so that the sample spreading layer would be outermost from the support, then would come the filtering layer, the reflecting layer, and finally the reagent layer.
An example of a layer which is useful as both a filtering layer and a reflecting layer, is a layer comprised of titanium dioxide or barium sulfate dispersed in a binder such as cellulose acetate, poly(vinyl alcohol), or gelatin. This layer is particu-larly useful in a multilayer tape intended for use in analysis of whole blood since it effectively screens out the blood cells while transmitting the serum and provides an effective white background for spectrophotometric measurements made through the support.
A particularly advantageous layer for use as a sample spreading layer in a multilayer tape intended for use in analysis of whole blood is a layer comprised of a dispersion of diatoma-ceous earth in a binder such as cellulose acetate. The diatoma-ceous earth is very effective in distributing the blood uniformly in a lateral direction. Sample spreading layers can also be pre-pared from microcrystalline colloidal products derived fromeither natural or synthetic polymeric materials. These micro-crystalline materials are described in an article entitled , . : ~ : ' 056Zl~Z
"Colloidal Macromolecular Phenomena, Part II, Novel Micro-crystals of Polymers" by O.A. Battista et al published in the Journal of Applied Polymer Scienee, Vol.II, pages 481-498 (1967).
Microcrystalline cellulose, which is commercially available from FMC Corporation under the trademark Avicel, is an example of a material of this type which is satisfactory for use in the present invention.
Good results are also obtained with a sample spreading layer comprised of inert spherical particles of uniform size held in a matrix of a binder material which bonds the particles to the underlying layer. Examples of such spherical particles are glass beads and polymeric resin beads. Gelatin and poly(vinyl alcohol) are particularly good binders for use with the glass beads or resin beads. The binder should be used in a small amount so as -to avoid filling any substantial portion of the void volume pro-vided by the spheres. A sample spreading layer comprised of spherieal partieles of uniform size provides advantages as eom-pared to a porous polymer layer such as a "blush polymer" layer.
Thus, it is effective in spreading a drop of blood, blood serum, etc., to a uniform and reproducible area and it does this so rapidly that the spreading is completed before any signifieant degree of diffusion of blood eomponents into adjaeent layers of the multilayer element ean oeeur. This results in a very uni-form concentration of eholesterol in the reagent layer and a uniform eolor or other eoneentration of indicator which is measured as a basis for the analysis. While blood cells can eause elogging of porous polymer layers, this does not oeeur with a sample spreading layer comprised of spherical partieles in a matrix of binder. The use of spheres of uniform size pro-vides espeeially desirable results as it provides an adequate volume of void spaee while limiting the average dimension of the interstitial spaees to a relatively narrow range. This results . .

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;~ in a cessation of spreading of the blood once the interstitial volume in the sample area has been completely filled and also permits rapid drainage of the plasma into the underlying layer~;
once the spreading process has been completed. Spheres of a size in the range of about 80 to 120 microns are particularly desirable for a sample spreading layer to be used with whole blood. A drop of blood placed on such a layer spreads in only a few seconds to a circle of uniform area and composition, and the area covered after spreading is directly proportional to the sample volume. The incorporation of a very small amount of a surfactant in the spreading layer, in addition to the spherical particles, is advantageous as it accelerates the spreading process.
Since the cholesterol oxidase in the reagent layer appears to offer at least in some cases resistance to binding of super-; imposed spreading, filtering and reflective layers, it has been found advantageous in some cases to apply a porous separating or :`
- interlayer which serves as a subbing layer to improve adhesion between the cholesterol oxidase containing layer and superimposed ~ayers. So long as the interlayer is sufficiently porous to permit the cholesterol to reach the reagent layer and provides the adhesion improvement desired, it may be formed of almost any material. Polymeric film forming materials are particularly useful in this application. Among those which have been found operative are:
poly(n-vinyl-2-pyrrolidone), poly(isopropylacrylamide), copoly(vinyl acetate-vinyl neodecanoate) (20 weight percent vinyl acetate), and copoly(vinyl neodecanoate-n-vinyl-2-pyrrolidone) (between 10 and 30 weight percent vinyl neodecanoate) Similar interlayers may be used to improve adhesion of the cholesterol oxidase containing layer and underlying layers, for ..~, example, indicator layers or to separate other portions of the reagent layer, or porous spreading medium, e.g., a hydrolysis layer as described below within the spreadiny medium. An espe-cially preferred polymeric material for use in this application is poly(n-isopropylacrylamide) applied from an acetone or acetone and water solution. Since it is critical that the porosity of the interlayer be maintained, these layers are necessarily very thin and may generally range in thickness from mono-layers of material on up to layers on the order of 1 mil. When polymeric interlayers of the materials mentioned above are used, these are generally applied at levels ranging from about 90 mg/m to about 1000 mg/m2 depending upon such properties as the density of the polymer, the porosity of the ultimate subbing layer, etc.

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In reading a blank analytical element of the type des-cribed herein, if, for example, in the reflection spectrophoto-metric mode, the photometer-detector sees only about 1% of the energy incident on the element, this 1% of incident radiation ; then becomes the 100% reference level for subsequent reflection measurements.
In typical measurements made with the same web structure, but with the photometer-detector moved just above the web, in what might be termed a "near total transmission" mode, the detector sees about 1.5 to 2% of the energy incident on the multilayer element. If the thickness of the reflective spreading layers as described hereinabove is reduced by about half, trans-mission rises to about 4%, and in elements using microcrystal-line cellulose spreading layers of thicknesses of from about 100 to about 300 microns, transmission will rise to nearly 20% of incident radiation. Thus, one has better signal-to-noise values in the transmission mode of detection, and it may be desirable in many cases to use this detection mode. Other layers in addi-tion to those which have been discussed above can also be ;.~,!, '.'''.' " ' `` 105628Z
included in the multilayer analytical element of this invention.
For example, a dialysis layer which is positioned directly over the reagent layer can be provided. A semipermeable cellulose membrane which serves to separate high molecular weight materials from low molecular weight materials would be suitable for a di-alysis layer. Such a layer functions, of course, by a different mechanism than a porous polymer layer, such as the "blush poly~
mer" layer described hereinabove, which merely acts as a screen in which the pores prevents the passage of particles which are too large to pass through them.
In analyzing whole blood with the analytical element of this invention, the blood cells may first be separated from the serum, by such means as centrifuging, and the serum applied to i~.
the tape. However, it is not necessary to make such separation, as whole blood can be applied directly to the element and the blood cells filtered out through the action of the filtering 7 layer if reflective spectrophotometric analysis techniques are used to quantify the reaction product formed in the element. The presence of these cells on the element will not interfere with 20 the spectrophotometric analysis since it is carried out by re-` flection techniques, with light being transmitted through the support and reagent layer and reflected from the porous reflecting layer. A particularly significant advantage of the analytical element described herein is its ability to be used to analyze either serum or whole blood. Of course, where energy transmis-sion techniques are used to quantify the amount of indicator formed in the element, the spreading layer and any other layers must be uniformly permeable to the detecting radiation, and unless some mechanism is provided for removing the undesired ~-' 30 residues of whole blood (e.g. wiping off or stripping off the spreading layer before measurement), it is preferable to use ; blood serum obtained in any conventional fashion to make the ~ " lOS6;~8Z

. . .
analysis. Furthermore, it is most desirable that whatever material is used for this layer when whole blood is applied there-to, that hemolysis of the red blood cells not occur since this would tend to give distorted assays which would reflect the presence of at least some intracellular cholesterol, if correction is made for this distortion, avoiding hemolysis is not so important.
The multilayer analytical elements of this invention can be manufactured in any appropriate width, a typical size being an :. .
~ element with a width of sixteen millimeters, and would typically ....
` 10 be produced in the form of a long length of tape wound on a spool .:, or enclosed in a casette. The use of individually mounted chips also provides a useful variation.
,~, : In the accompanying drawing, Figures 1 to 5 are enlarged sectional views of preferred embodiments of the multilayer analy-tical elements of the present invention. As shown in Figure 1, an analytical element is composed of a support 10, on which is '~'I .
coated a reagent layer 12, a reflecting layer 14, which provides a white background for reflection spectrophotometry through sup-port 10, a filtering layer 16, and a sample spreading layer 18.
,.;' Reagent layer 12 can be composed of a dispersion of one or more ~ test reagents in a binder such as gelatin, while each of layers ;' 14, 16, and 18 can be a "blush polymer" layer having a pore size ' adapted to the particular function it is intended to perform. The ' specific components of the reagent layer are described hereinafter.
In an alternative embodiment of the invention shown in Figure 2, the analytical element is composed of a support 20 bearing a rea-; gent layer 22 and a layer 24 which serves the function of sample spreading and filtering, and which also may provide a suitable background for reflection spectrophotometry through support 20.
Alternatively, layer 24 may be such that it does not reflect and quantization is accomplished in the transmission mode. Layer 24 can be, for example, a "blush polymer" layer which has been . : ~ - :, lOX6Z8Z

coated over layer 22 or a layer of a microporous filter membrane `~ which has been laminated to layer 22. Figure 3 illustrates a further embodiment of the invention in which the analytical ele-` ment is composed of support 30, reagent layer 32, a dialysis layer 34, which is formed from a semi-permeable membrane, and a layer 36, such as a "blush polymer" layer, which serves the func-tions of sample spreading and filtering, and which provides a ~-suitable background for reflection spectrophotometry through sup-port 30. A still further embodiment of the invention is shown in Figure 4 in which the analytical element is composed of support 40, a first reagent layer 42, a second reagent layer 44, a layer 46, ; which serves as a filtering and light reflecting layer, and a :
sample spreading layer 48. Layer 46 can be composed, for example, . ~
of a dispersion of titanium dioxide in cellulose acetate and layer 48 can be composed of a dispersion of diatomaceous earth in cellulose acetate or of glass beads in gelatin.
:
As shown in Figure 5, according to a highly preferred em-bodiment, the support 50 is coated with a first reagent layer 52 ~ which includes the color indicator system as described below dis- -; 20 persed in a suitable matrix material coated over first reagent layer 52 and a "barrier" layer 54 whose purpose and composition are described in detail hereinafter. Over the barrier layer are second reagent layer 56 which contains the components of the cho-~ lesterol ester hydrolysis system, i.e., the lipase preparation ; which demonstrates cholesterol esterase activity and the protease, and the cholesterol oxidase. A combined spreading, filtering and reflecting layer of 58 of a composition similar to layers 46 and 48 described above, is coated over the second reagent layer.
As all of the layers described herein are formed by coating from solutions or dispersions as described in the aforementioned Belgian Patent No. 801,742, it is often '`:

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necessary to include coating aids which impart uniform coating properties to the layers.
Whatever coating aids are used for this purpose, or those described below, it is important that they do not inhibit the lipase or any of the other reagents present in any of the various reagent layers. Particularly useful coating aids for this purpose include nonionic surfactants such as the octyl phenoxy polyethoxy ethanols commercially available from Rohm and Haas Co. under the Triton~ tradename (X-100, 102, 165, 305 and 405 being particularly useful), (p-nonylphenoxy) glycerol commercially available from Olin Mathieson Corp. under the tradename Surfactant 10 ~, and polyethylene glycols such as the Carbowax~ materials available from Union Carbide.
Furthermore, although the coating aids serve to impart uniform, desirable coating characteristics to the various layers, it is particularly important that the reagent layer which includes the cholesterol oxidase also include a concentration of from about .5 to about 5 g/m2 and preferably from about 1 to about 3 g/m2 of a surfactant to insure proper oxidation of the free cholesterol by the oxidase enzyme. Although some reaction will occur without this concentration of surfactant, useful quantitative results can only be achieved when it is present. Concentrations of surfactant above 5 g/m2 cause degradation of the physical properties of the web. Optimum quantitative results have been obtained when the coating aid or surfactant is a nonyl phenoxy polyethoxy ethylene of the type commercially available from Rohm and Haas under the tradename Triton X-100~.
The reagent system of the preferred embodiment of the present invention can be looked at very basically as a three part composite. Two portions of the composite can be eliminated to provide somewhat less desirable, however, very useful, alternative 10562~3Z

embodiments depending upon the character of the sample under analysis and the quantization technique to be utilized. For example, if fluorescent quantization of cholest-4-ene-3-one is : used as the indicator, the color indicator system may be omitted.
Similarly, if the web is to be spotted with solutions containing only "free" cholesterol, the enzymatic hydrolysis system may be deleted.
The chemical reactions involved in a preferred total pro-cess of this invention are as follows:

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3 ~:

Reaction (1) indicates the release of free cholesterol from cholesterol and cholesterol-esters complexed with serum lipo-proteins. Equation (2) shows the cholesterol oxidase reaction.
Reaction (3) demonstrates one of the many possible dye-peroxidase systems which may be used to detect H2O2 production according to a preferred embodiment of the invention. Here a system involving oxidation of 4-aminoantipyrine and 1,7-dihydroxynaphthalene to a compound with an absorption maximum at 490nm is shown. This dye system is chosen because of its sensitivity, its stability, and the lack of interference by other serum components. Blanks, run concomitantly with samples but lacking the oxidase, give no sig-nificant change in optical density within the times chosen for the assay as specified below. Of course, as mentioned above and described in greater detail below, any number of quantitating sys-tems may be used in the successfulpractice of the invention.
The foundation stone of the entire reagent system of the ;
instant invention is the cholesterol oxidase enzyme. This enzyme, which catalyzes the oxidation of cholesterol to cholest-4-ene-3-one and hydrogen peroxide in the presence of oxygen, is the principal reagent which permits the application of a fluid con-taining cholesterol and cholesterol esters to a dry web, and the direct photometric or fluorometric reading of cholesterol concen-tration from that web.
As alluded to hereinabove, the synthesis of cholesterol oxidase is described in detail in Goodhue and Risley U. S. Patent No. 3,909,359. Basically, such a synthesis comprises growing the bacterium Nocardia cholesterolicum species NRRL 5767 or NRRL
5768 in a conventional growth medium comprising a substrate, an ammonia source, a potassium source, a phosphorus source, trace metal ions, and a carbon source, and isolating from such mixture :

` 1056282 using well-known techniques, a cell free extract containing the active enzyme. A crude technique for preparing this enzyme is described in Stadtman, T. C., Methods in Enzymology, Vol. 1, Colowick, S. P. and Kaplan, N. O., Eds. Academic Press, N. Y., 1955, p. 678, and Stadtman, T. C., Cherkes, A. and Anfinsen, J., Biol. Chem., 206, 511 (1954). According to the preferred embodi-ment described in the aforementioned U. S. Patent No. 3,909,359 the enzyme synthesis is accomplished in the presence of a primary carbon source such as glycerol and an inducer selected from the group consisting of cholesterol, cholesteryl linoleate, and cholest-4-ene-3-one. The preparation of a distinctly different cholesterol oxidase (based upon the published morphology of the bacteria used to produce the enzyme and on physical chemical characteristics of the enzymes) is described in German Offenlegungsschrift 2,246,695 published March 26, 1973. This technique involves the growth of Nocardia species NRRL 5635 or 5636 according to the procedures described in the subject German patent publication. Dispersion of either of these materials in a reagent binder of the type described above using conventional techniques provides a useful reagent layer.
Since cholesterol oxidase, as most enzymes, operates efficiently only within a relatively narrow pH range, it is gen-erally necessary to obtain an efficient element to buffer the reagent layer containing this enzyme at some pH value within the operative pH range of the enzyme. Thus, although it is possible to detect some enzymatic activity outside of this range, it is desirable to buffer the reagent layer containing the cholesterol oxidase between about 5.5 and 8.5 and preferably between about 6.o and 7Ø Techniques for achieving this type of buffering are well known in the art and involve dissolving or dispersing the buffering agent in the reagent system prior to coating. Suitable buffering agents for buffering to the aforementioned pH are ~r. ~, 1056Z8iZ
described in detail by Good in Biochemistry 5, 467 (1966). Par-ticularly useful buffers include the phosphates such as potassium phosphate, the so-called Tris ~i.e., tris(hydroxymethyl)amino-methane~ and HEPES (i.e., N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) buffers and dimethyl glutarate. ;
The action of oxygen on free cholesterol in the presence ; of cholesterol oxidase produces hydrogen peroxide and cholest-

4-ene-3-one. Thus, if a solution containing free cholesterol is to be analyzed, a reagent layer containing only a dispersion of cholesterol oxidase in gelatin at a concentration of between about 0.01 and about 0.5 units per square centimeter and prefer-ably between about 0.05 and about 0.2 units per square centimeter could be used since cholest-4-ene-3-one fluoresces at 290 nm and the concentration thereof could be measured simply by direct fluorescence measurements. Similar concentrations of cholesterol oxidase are useful when the complete hydrolysis and indicator systems are also incorporated into the web. This concentration of enzyme may, of course, be varied over a broad range and very limited experimentation will permit the skilled artisan to determine optimum levels for his particular element.
According to a preferred embodiment of the present inven-tion, however, cholesterol quantization in complex mixtures con-taining both free and esterified cholesterol is achieved using an indicator system which quantifies the level of hydrogen peroxide generated in the oxidation of cholesterol. Indicator systems for the detection of enzymatically generated hydrogen peroxide are well known in the art particularly as indicator systems in enzyma-tic glucose and uric acid detection elements and techniques. U.S.
Patent Nos. 3,092,465 and 2,981,606 describe quantitating composi-tions which are useful in the successful practice of the presentinvention. The hydrogen peroxide indicator systems generally comprise a substance having peroxidative activity, preferably , . .

peroxidase as defined in the aforementioned references, and an indicator material which undergoes a color formation or change in the presence of hydrogen peroxide and oxygen. Alternatively, the indicator material may be one or more substances which undergo no substantial color change upon oxidation in the presence of H2O2 and peroxidase, but which in their oxidized form react with a color-forming or -changing substance to give visible quantita-tive evidence of chemical reaction. U.S. Patent No. 2,981,606 in particular provides a detailed description of such color indicator systems. The latter color forming system, i.e. one which pro-duces color by virtue of an intermediate or color coupling re-action is preferred in the practice of the present invention.
Such a system involves incorporating either into the reagent layer containing the cholesterol oxidase or another contiguous or sepa-rated stratum of the reagent layer the components of the color or other energy absorbing or emitting indicator system. This can be accomplished merely by dispersing the components of the indicator system described below into a reagent layer binder of the type described above, preferably gelatin and coating as described in 20 Belgian Patent No. 801,742, issued January 2, 1974, cited above.
A peroxidase is an enzyme which will catalyze a reaction wherein hydrogen peroxide oxidizes another substance. The per-oxidases are generally conjugated proteins containing iron por-phyrin. Peroxidase occurs in horseradish, potatoes, figtree sap and turnips (plant peroxidase); in milk (lacto peroxidase); and in white blood corpuscles (verdo peroxidase); also it occurs in microorganisms. Certain synthetic peroxidases, such as disclosed by Theorell and Maehly in Acta Chem. Scand., Vol. 4, pages 422-434 (1950), are also satisfactory. Less satisfactory are such substances as hemin, methemoglobin, oxyhemoglobin, hemoglobin, hemochromogen, alkaline hematin, hemin derivatives, and certain other compounds which demonstrate peroxidative or peroxidase-like activity, namely, the ability to catalyze the oxidation of 1~56Z8Z
another substance by means of hydrogen peroxide and other perox-ides. The various peroxidases are believed to contain hematin.
Other substances which are not enzymes but which possess peroxidase-like activity are: iron sulfocyanate, iron tannate ferrous ferrocyanide, chromic salts (such as potassium chromic sulfate) absorbed in silica gel, etc. These substances are not as satisfactory as peroxidase per se.
Color-forming substrates of peroxidase and peroxidase-like substances which produce a color formation in the presence of hydrogen peroxide and peroxidase which may be employed in the indicator of the present invention include the following sub-stances with a coupler where necessary:
(1) Monoamines, such as aniline and its derivatives, ortho-toluidine, para-toluidine, etc.;
(2) Diamines,such as ortho-phenylenediamine, N,N'-dimethyl-para-phenylenediamine, N,N'-diethyl phenylenediamine, benzidine (which produces a blue or brown color), dianisidine (turns green or brown), etc.;
(3~ Phenols, such as phenol per se (producing a yellow color), thymol, ortho-, meta and para-cresols (producing a green-yellow color, a pink color and a milky suspension, respectively), alpha-naphthol (producing a magenta color), beta-naphthol (pro-ducing a white precipitate), etc.;
(4) Polyphenols, such as catechol, guaiacol (which forms an orange color), orcinol, pyrogallol (producing a reddish or yellow color), p,p-dihydroxydiphenyl and phloroglucinol;

(5) Aromatic acids, such as salicyclic, pyrocatechuic and gallic acids;

(6) Leuco dyes, such as leucomalachite green (to pro-duce malachite green) and leucophenolphthalein (desirably employedin an alkaline medium);

(7) Colored dyes, such as 2,6-dichlorophenolindophenol;

:.: ' -' ... .

1056~Z~Z

(8) Various biological substances, such as epinephrine, the flavones, tyrosine, dihydroxyphenylalanine (producing an orange-reddish color) and tryptophan;

(9) Other substances, such as gum guaiac, guaiaconic acid, potassium, sodium, and other water soluble iodides; and bilirubin (producing a greenish color); and

(10) Such particular dyes as 2,2'-azine-di(3-ethylbenzo-thiazoline-(6)-sulfonic acid) and 3,3'-diaminobenzidine.
The color indicator system of the present invention preferably comprises 4-methoxy-1-naphthol which undergoes self coupling in its oxidized state or a combination of 1-7 dihydroxy-naphthalene and 4-aminoantipyrine (HCl). In the latter system the oxidized pyrine compound coupleswith the naphthalene. The concentrations of the various color indicator systems useful in the elements described herein are dependent to large extent upon the concentration of cholesterol in the sample, the sophistication of the detection apparatus, etc. and are readily determinable by the skilled artisan. Typical valuesare shown in the examples below.
As mentioned above, the optimum element of the present invention also includes a hydrolysis medium which saponifies any cholesterol esters present in the sample to "free" cholesterol.
Such a hydrolysis system is described in detail in U. S. Patent No. 3,869,349. This hydrolysis system comprises a lipase having esterase activity and a protease. Generally, this method requires treating the serum with a mixture of enzymes comprising a lipase having esterase activity and a protease. This combination of enzymes quite unexpectedly saponified the cholesterol esters in a highly efficient manner.
A number of lipases hydrolyze cholesterol esters to some degree as described in the aforementioned patent~

A preferred lipase for the analytical solutions of the present invention is Lipase M from Candida cylindracca marketed by Enzyme Development Co., which provides quantitative hydrolysis of serum cholesterol esters in a period on the order of 10 minutes at 50C.
To obtain maximum effect from the lipase, as described in detail in the aforementioned Goodhue et al application, it is necessary to incorporate a protease. Several microbial proteases are satis-factory including bromelain and the proteases from Streptomyces griseus and Bacillus subtilis.
A useful screening technique for determining the esterase activity of lipase enzymes is described in detail in Example 3 of the aforementioned U. S. Patent No. 3,869,349 and generally com-prises adding a fixed amount of a lipase preparation to a standard cholesteryl linoleate solution at pH 7.0, incubating at 37C. ~ ~ -under N2 for 2 hours and determining the amount of ester left in the solution by the hydroxylamine method of J. Vonhoeffmayr and R. Fried, Z. Klin. Chem. U. Klin. Biochem., 8, 134 (1970).
The lipase present in the test solution of the instant invention may be of plant or animal origin but must demonstrate esterase activity as described hereinabove. Among the useful lipases it is preferred to use a microbial lipase such as the lipase from Candida cylindracca and lipases having similar activity. Specifically preferred commercial lipases include wheat germ lipase supplied by Miles Laboratories of Elkhart, Indiana, Lipase 3000 supplied by Wilson Laboratories, Steapsin~ (both of the former are pancreatic enzymes) supplied by Sigma Chemical Co., and Lipase ~ (from Candida cylindracca) supplied by Enzyme Development Co. Screening of lipasesfor this purpose to determine their cholesterol esterase activity may be accomplished using the technique referred to in the preceeding paragraph. Using this tech-nique, any lipase which demonstrates a cholesterol esterase activity - - . . :

:: ' lOS6282 which releases above about 25 mg% cholesterol in the screening procedure should be considered useful in the practice of the present invention.
Proteases in general may be used. These include by way of examples, chymotrypsin, Streptomyces griseus protease (com-merically available under the registered trademark "Pronase"), proteases from Aspergillus oryzae, Bacillus subtilis, elastase, papain, and bromelain. Mixtures of such enzymes may of course also be employed.
Such a hydrolysis system may be incorporated into the cholesterol oxidase reagent layer described immediately herein-above, however, according to a highly preferred embodiment of the present invention, the hydrolysis medium is incorporated into the porous spreading layer simply by dispersing the enzymes in a lyophilized state in the coating medium used to form the spread-ing layer, and then coating this mixture over the reagent layer as described in Belgian Patent 801,742. According to this embodi-ment, spreading of the sample and hydrolysis of any cholesterol esters are accomplished simultaneously and the sample reaches the reagent layer all in the form of free cholesterol thereby utiliz-ing the time used to spread and transmit the sample to simul-taneously prepare it for total and immediate reaction with the cholesterol oxidase in the main stratum of the reagent layer.
Alternatively, of course, a distinct contiguous or separated rea-gent stratum may be incorporated into the reagent layer between the spreading layer and the cholesterol oxidase stratum to accom-plish hydrolysis before the sample reaches the cholesterol oxidase.
Whenever the enzymatic cholesterol hydrolysis system is incorporated, optimum results are achieved when the matrix is buffered to a pH of between about 5 and 9.5 and preferably be-tween about 7.0 and 8Ø Thus, when the hydrolysis system is incorporated into the reagent, or in another layer with the cholesterol oxidase, a pH of about 7.0 produces optimum results.

Similar pH's are used when the hydrolysis system is used in a second reagent layer as described below.
The concentration of lipase and protease in whatever layer the hydrolysis system is incorporated may vary over a broad range. Generally, however, concentrations of lipase ranging from about 90,000 to about 270,000U/m2 and protease ranging from about 36,000 to about 105,000U/m2 have been found useful. selow the levels expressed immediately hereinabove substantially complete i hydrolysis is doubtful. Concentrations of these components above these levels, although perhaps useful, are not commercially attractive. According to a preferred embodiment of the present invention, lipase levels on the order of from about 150,000 to ~ -about 200,000 U/m2 and protease concentration of from about 72,000 to about 90,000 U/m are used.
As one might expect, the incorporation of a protease into - -; a reagent layer whose matrix is composed primarily of gelatin poses certain stability problems since the protease attacks the protenaceous gelatin as soon as wetting of the mixture occurs.
Although some measurements can be made in an element which in-cludes the protease and consequently the hydrolysis system in the gelatin reagent layer, it is most desirable that the hydrolysis system be incorporated into the polymeric binder of the spreading, filtering, and reflecting layer, which is resistant to the action - of the protease and that, as a further measure to protect the gelatin matrix of the first reagent layer from the protease, that a protective barrier layer of the type depicted at 54 in Figure 5 be incorporated into the element. In this configuration, it is also desirable to place the cholesterol oxidase in close -proximity to the hydrolysis system so that indication in first reagent layer 52 requires only that the relatively small hydrogen `

peroxide molecules be permitted to cross the barrier layer while the larger protease enzyme molecules are prohibited lOS6Z82 from migrating between the first and second reagent layers.
Barrier layers of the type described herein are, however, equally useful for the purpose of protecting reagent layer compositions which contain the cholesterol oxidase with or without an indicator system as they permit passage of free cholesterol from the por-ous spreading medium to the reagent layer while prohibiting passage of the protease.
The barrier layer may be comprised of any of a large variety of materials compatible with the various components of the web. However, generally preferred barrier layers are com-prised of hydrophilic polymeric materials which permit migration of the hydrogen peroxide or free cholesterol as just described while at the same time excluding the protease enzyme and demon-strating no inhibitory effect on any of the other components of the system. Particularly preferred as the protective barrier layer is a coating of agarose at a coverage ranging from about 0.1 to 1 g/m . A highly preferred embodiment of the present invention utilizes a layer of agarose at a coverage of from about 0.25 to about 0.70 g/m .
The following examples will serve to better illustrate the successful practice of the present invention.
Example 1 An analytical element containing all the necessary rea-gents for the quantitative analysis of cholesterol, in blood serum, is prepared in the following manner. A sample of a gelatin subbed 7 mil. poly(ethylene terephthalate) film support is coated with an indicator layer comprising gelatin (21.5 g/m2), peroxidase (7,000 U/m2), 4-methoxy-1-naphthol (750 mg/m2), bis(vinylsulfonyl-methyl) ether (129 mg/m2) and phosphate buffer to pH 6.93. The above described indicator layer is then overcoated with a reagent layer comprising gelatin (5.56 g/m2), octyl phenoxy polyethoxy ethanol(l70mg/m2), cholesterol oxidase(54 U/m2), and phosphate buffer .
to pH 7.0 An interlayer comprising poly (n-isopropylacrylamide) (540 mg/m ) is then applied to the element followed by a spreading layer comprising cellulose acetate (9.7 g/m2), and titanium dioxide (64.5 g/m2).
To evaluate the coated element a series of cholesterol standards varying in concentration from 50 to 400 mg% were pre-pared by dissolving cholesterol in Gafac~ L0-529 (a sodium salt of complex organic phosphate esters, available from GAF Corpora-tion, Dyestuff and Chemical Division).
The coating was spotted with 10 ~1 drops of the above , described cholesterol solutions, a spectrophotometer at 37C. with a 660 nm infrared filter was used to follow color development at times varying from 5-20 minutes. The results were recorded and are plotted in Figure 6.
Example 2 An analytical element containing all the necessary reagents for the quantitative analysis of total cholesterol, in blood serum, is prepared in the following manner. A sample of a gelatin subbed 7 mil. polyethylene terephthalate) film support is coated with an indicator layer comprising gelatin (21.5 g/m2) peroxldase (7,000 U/m2), bis(vinylsulfonylmethyl) ether (430 mg/m2), cholesterol oxidase (1,936 U/m2), octylphenoxypolyethoxy ethanol (Triton X-100, 2.7 g/m2), 4-methoxy-1-naphthol (750 mg/m2), 5,5-dimethyl-1,3-cyclohexane dione (215 mg/m2) and phosphate buffer to pH 6.43. An interlayer comprising poly(n-isopropylacrylamide) (323 mg/m2) is then applied followed by a spreading layer com-prising cellulose acetate (9.7 g/m2), titanium dioxide (64.5 g/m2), Lipase M (1.08 g/m2), ~-chymotrypsin (2.15 g/m2) and Triton X-100 (2.96 g/m2).
To evaluate the coated element a series of blood serum samples containing 122, 244 and 366 mg% cholesterol are applied to the coated element (10 ~1 drops), after 12 minutes at 37C.

33 ~

, . , ' ' . - ..

; 1056Z82 a spectrophotometer with a 660nm IF is used to measure the reflec-tion density (DR) of the element, with the following results.
Test Serum DR660nm (12 min. at 37 C
_ 122 0.12 244 0.18 366 0.19 Example 3 An analytical element containing all the necessary rea-gents for the quantitative analysis of total cholesterol, in blood serum is prepared in the following manner. A sample of gelatin subbed 7 mil. poly(ethylene terephthalate) film support is coated with an indicator layer comprising gelatin (21.5 g/m2), peroxidase (7,000 U/m2), cholesterol oxidase (430 U/m2), 1,7-dihydroxy naph-thalene (656 mg/m2) 4-aminoantipyrine hydrochloride (635 mg/m2) and 4-amino-5,6-dihydroxy-2-methylpyrimidine (10.8 mg/m2) at a pH
of 7Ø A barrier layer comprising Agarose (108 mg/m2) was then applied followed by an interlayer comprising poly(n-isopropylacryl-amide) (323 mg/m2) and a spreading layer containing hydrolysis enzymes as described in Example 2.
Upon evaluation, as in Example 2, comparable results were obtained.
Example 4 An analytical element containing all the necessary rea-gents for the quantitative analysis of total cholesterol, in blood serum, is prepared exactly as in Example 3 with the following exceptions.
(1) The indicator layer contained 4-methoxy-1-naphthol (750 mg/m ) instead of 1,7-dihydroxy naphthalene and 4-aminoantipyrine.
(2) The cholesterol oxidase was removed from the indicator layer and coated in the spreading layer (450 U/m2).

Upon evaluation, as in Example 2, the following results were obtained.
Test Serum DR 660nm (12 min. at 37C

122 0.12 244 0.21 366 0.31 Example 5 An analytical element containing all the necessary rea- -~
gents for the quantitative analysis of total cholesterol, in blood serum, is prepared exactly as Example 3 except the cholesterol oxidase was removed from the indicator layer and coated in the spreading layer (450 U/m2).
Upon evaluation, as in Example 2, results comparable to those of Example 4 were obtained.
The results of these tests demonstrate the response of the analytical element of the present invention to cholesterol standards.
While the invention has been described in detail with particular reference to preferred embodiments thereof, it will be understood that variations and modifications can be effected within the spirit and scope of the invention.

- ..

Claims (74)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A multilayer analytical element for use in the quanti-tative assay of complex fluids applied thereto for cholesterol content comprising: (1) a liquid-impermeable, radiant energy-transmitting support; (2) a reagent layer on one side of said support said reagent layer comprising a substance having cholester-ol oxidase activity dispersed in a binder; and (3) coated over said reagent layer a porous medium comprised of one or more layers, through which cholesterol present in said complex fluid is trans-mitted; said porous medium being adapted to spread the complex fluid in a manner providing a substantially constant volume of sample per unit area to the reagent layer.

2. A multilayer analytical element as described in claim 1 wherein said liquid-impermeable support transmits energy of a wavelength of between about 200 and about 900 nm.

3. A multilayer analytical element as described in claim 2 wherein said substance having cholesterol oxidizing activity comprises a cholesterol oxidase enzyme.

4. A multilayer analytical element as described in claim 3 wherein said cholesterol oxidase enzyme is derived from a microorganism selected from the group consisting of NRRL 5635, NRRL 5636, NRRL 5767 and NRRL 5768.

5. A multilayer analytical element as described in claim 4 wherein said microorganism is selected from the group consisting of NRRL 5767 and NRRL 5768 and said reagent layer is buffered at a pH of between about 5.5 and 7.5.

6. A multilayer analytical element as described in claim 5 wherein said reagent layer is buffered at a pH of between about 6.0 and 7Ø

7. A multilayer analytical element as described in claim 3 wherein said reagent layer also includes an indicator system which reacts with one or more products of the cholesterol oxidase induced decomposition of cholesterol in said complex fluid to produce a spectrophotometrically quantizable energy absorption shift.

8. A multilayer analytical element as described in claim 7 wherein said indicator system comprises a substance having peroxidative activity and a composition which undergoes a color change in the presence of hydrogen peroxide and said substance demonstrating peroxidative activity.

9. A multilayer analytical element as described in claim 8 wherein said substance having peroxidative activity is a peroxidase enzyme.

10. A multilayer analytical element as described in claim 9 wherein said composition which undergoes a color change in the presence of hydrogen peroxide and peroxidase is a leuco dye.

11. A multilayer analytical element as described in claim 9 wherein said composition which undergoes a color change in the presence of hydrogen peroxide and peroxidase comprises a substance which is oxidized in the presence of hydrogen peroxide and oxygen without itself undergoing a color change, but which in its oxidized state is capable of coupling to form a dye, with itself or a coupler capable of reacting with said oxidized sub-stance.

12. A multilayer analytical element as described in claim 11 wherein said substance which is capable of coupling with itself in its oxidized state is 4-methoxy-1-naphthol.

13. A multilayer analytical element as described in claim 11 wherein said substances which is oxidized in the presence of hydrogen peroxide and peroxidase without itself undergoing a color change is 4-aminoantipyrine.

14. A multilayer analytical element as described in claim 13 wherein said coupler is 1,7-dihydroxy naphthalene.

15. A multilayer analytical element as described in claim 3 wherein said reagent layer includes a lipase having cholesterol esterase activity and a protease which combination of enzymes serves to hydrolyze cholesterol esters which may be present in said complex fluid.

16. A multilayer analytical element as described in claim 15 wherein said lipase having esterase activity releases at least 25 mg% cholesterol in 2 hours at 37°C under nitrogen when 50 mg of a preparation of said lipase in 5 ml 0.1 M phos-phate buffer, pH 7.0, is used to treat a dispersion of cholesteryl linoleate prepared by dispersing 200 mg cholesteryl linoleate in 5 ml of ethyl ether and 100 ml boiling water containing 430 mg of sodium cholate.

17. A multilayer element as described in claim 15 wherein the concentration of said lipase in said reagent layer ranges from about 90,000 to about 270,000 U/m2 and the concen-tration of said protease in said reagent layer ranges from about 36,000 to about 105,000 U/m2.

18. A multilayer analytical element as described in claim 15 wherein said reagent layer is buffered to a pH of between about 5.5 and about 8.5.

19. A multilayer analytical element as described in claim 17 wherein said lipase is a microbial lipase.

20. A multilayer analytical element as described in claim 19 wherein said lipase is from Candida cylindracca.

21. A multilayer analytical element as described in claim 17 wherein said lipase is selected from the group con-sisting of wheat germ lipase, pancreatic lipases and the lipase from Candida cylindracca.

22. A multilayer analytical element as described in claim 15 wherein said protease is selected from the group con-sisting of Bacillus subtilis protease, Streptomyces griseus protease, Aspergillus oryzae protease and mixtures thereof.

23. A multilayer analytical element as described in claim 22 wherein said protease is Bacillus subtilis protease.

24. A multilayer analytical element as described in claim 3 wherein said porous medium includes a lipase having cho-lesterol esterase activity and a protease which combination of enzymes serves to hydrolyze cholesterol esters which may be present in said complex fluid.

25. A multilayer analytical element as described in claim 24 further including a barrier layer between said porous medium and said reagent layer which barrier layer prohibits passage of said protease to said reagent layer while permitting free cholesterol to reach said reagent layer.

26. A multilayer analytical element as described in claim 25 wherein said barrier layer comprises agarose.

27. A multilayer analytical element as described in claim 25 wherein said lipase having esterase activity releases at least 25 mg% cholesterol in 2 hours at 37°C under nitrogen when 50 mg of a preparation of said lipase in 5 ml 0.1 M phos-phate buffer, pH 7.0, is used to treat a dispersion of cholesteryl linoleate prepared by dispersing 200 mg cholesteryl linoleate in 5 ml of ethyl ether and 100 ml boiling water containing 430 mg of sodium cholate.

28. A multilayer element as described in claim 24 wherein the concentration of said lipase in said reagent layer ranges from about 90,000 to about 270,000 U/m2 and the concen-tration of said protease in said reagent layer ranges from about 36,000 to about 105,000 U/m2.

29. A multilayer analytical element as described in claim 24 wherein said reagent layer is buffered to a pH of between about 5.5 and about 8.5 and said porous medium is buffered to a pH of between about 5 and 9.5.

30. A multilayer analytical element as described in claim 28 wherein said lipase is a microbial lipase.

31. A multilayer analytical element as described in claim 30 wherein said lipase is from Candida cylindracca.

32. A multilayer analytical element as described in claim 28 wherein said lipase is selected from the group con-sisting of wheat germ lipase, pancreatic lipases and the lipase from Candida cylindracca.

33. A multilayer analytical element as described in claim 24 wherein said protease is selected from the group con-sisting of Bacillus subtilis protease, Streptomyces griseus protease, Aspergillus oryzae protease and mixtures thereof.

34. A multilayer analytical element as described in claim 33 wherein said protease is Bacillus subtilis protease.

35. A multilayer analytical element for use in the quantitative assay of complex fluids applied thereto for choles-terol content comprising:
(1) a liquid impermeable radiant energy-transmitting support;
(2) a reagent layer coated on one side of said support; and (3) coated over said reagent layer a porous medium com-prised of one or more layers adapted to spread the com-plex fluid in a manner providing a substantially con-stant volume of sample per unit area to the reagent layer and having dispersed therein a cholesterol oxidase enzyme and a cholesterol ester hydrolysis system comprising a lipase having cholesterol esterase activity and a protease.

36. A multilayer analytical element as described in claim 35 wherein said liquid-impermeable support transmits energy of a wavelength of between about 200 and about 900 nm.

37. A multilayer analytical element as described in claim 36 wherein said porous medium also serves the function of reflecting light transmitted through the support when quantifi-cation is carried out using reflective spectrophotometric analysis through the base.

38. A multilayer analytical element as described in claim 37 wherein said cholesterol oxidase enzyme is derived from a microorganism selected from the group consisting of NRRL 5635, NRRL 5636, NRRL 5767 and NRRL 5768.

39. A multilayer analytical element as described in claim 38 wherein said microorganism is selected from the group consisting of NRRL 5767 and NRRL 5768 and said porous medium layer is buffered at a pH of between about 5.5 and 8.5.

40. A multilayer analytical element as described in claim 39 wherein said porous medium layer is buffered at a pH
of between about 6.0 and 7Ø

41. A multilayer element as described in claim 37 wherein the concentration of said lipase in said reagent layer ranges from about 90,000 to about 270,000 U/m2 and the concen-tration of said protease in said reagent layer ranges from about 36,000 to about 105,000 U/m2.

42. A multilayer analytical element as described in claim 37 wherein said porous medium layer is buffered to a pH
of between about 5.5 and about 8.5.

43. A multilayer analytical element as described in claim 37 wherein said lipase is a microbial lipase.

44. A multilayer analytical element as described in claim 43 wherein said lipase is from Candida cylindracca.

45. A multilayer analytical element as described in claim 37 wherein said lipase is selected from the group consisting of wheat germ lipase, pancreatic lipases and the lipase from Candida cylindracca.

46. A multilayer analytical element as described in claim 37 wherein said protease is selected from the group con-sisting of Bacillus subtilis protease, Streptomyces griseus protease, Aspergillus oryzae protease and mixtures thereof.

47. A multilayer analytical element as described in claim 46 wherein said protease is Bacillus subtilis protease.

48. A multilayer analytical element as described in claim 37 wherein said reagent layer also includes an indicator system which reacts with one or more products of the cholesterol oxidase induced decomposition of cholesterol in said complex fluid to produce a spectrophotometrically quantizable energy absorption shift.

49. A multilayer analytical element as described in claim 48 wherein said indicator system comprises a substance having peroxidative activity and a composition which undergoes a color change in the presence of hydrogen peroxide and said substance demonstrating peroxidative activity.

50. A multilayer analytical element as described in claim 49 wherein said substance having peroxidative activity is a peroxidase enzyme.

51. A multilayer analytical element as described in claim 50 wherein said composition which undergoes a color change in the presence of hydrogen peroxide and peroxidase is a leuco dye.

52. A multilayer analytical element as described in claim 50 wherein said composition which undergoes a color change in the presence of hydrogen peroxide and peroxidase comprises a substance which is oxidized in the presence of hydrogen peroxide and oxygen without itself undergoing a color change, but which in its oxidized state is capable of coupling to form a dye, with itself or a coupler capable of reacting with said oxidized sub-stance.

53. A multilayer analytical element as described in claim 51 wherein said substance which is capable of coupling with itself in its oxidized state is 4, methoxy-1-naphthol.

54. A multilayer analytical element as described in claim 52 wherein said substance which is oxidized in the presence of hydrogen peroxide and peroxidase without itself undergoing a color change is 4-aminoantipyrine (HCl).

55. A multilayer analytical element as described in claim 54 wherein said coupler is 1,7-dihydroxynaphthalene.

56. A multilayer analytical element as described in claim 37 further including a barrier layer between said porous medium and said reagent layer which barrier layer prohibits passage of said protease to said reagent layer while permitting free cholesterol to reach said reagent layer.

57. A multilayer analytical element as described in claim 56 wherein said barrier layer comprises agarose.

58. A multilayer analytical element as described in claim 37 wherein said reagent layer also includes an indicator system which reacts with one or more products of the cholesterol oxidase induced decompostion of cholesterol in said complex fluid to produce a spectrophotmetrically quantizable energy absorption shift.

59. A multilayer analytical element as described in claim 58 wherein said indicator system comprises a substance having peroxidative activity and a composition which undergoes a color change in the presence of hydrogen peroxide and said substance demonstrating peroxidative activity.

60. A multilayer analytical element as described in claim 59 wherein said substance having peroxidative activity is a peroxidase enzyme.

61. A multilayer analytical element as described in claim 60 wherein said composition which undergoes a color change in the presence of hydrogen peroxide and peroxidase is a leuco dye.

62. A multilayer analytical element as described in claim 60 wherein said composition which undergoes a color change in the presence of hydrogen peroxide and peroxidase comprises a substance which is oxidized in the presence of hydrogen peroxide and oxygen without itself undergoing a color change, but which in its oxidized state is capable of coupling to form a dye, with itself or a coupler capable of reacting with said oxidized sub-stance.

63. A multilayer analytical element as described in claim 62 wherein said substance which is capable of coupling with itself in its oxidized state is 4-methoxy-1-naphthol.

64. A multilayer analytical element as described in claim 62 wherein said substances which is oxidized in the presence of hydrogen peroxide and peroxidase without itself undergoing a color change is 4-aminoantipyrine (HCl).

65. A multilayer analytical element as described in claim 64 wherein said coupler is 1,7-dihydroxynaphthalene.

66. A multilayer analytical element as described in claim 37 wherein said reagent layer comprises a binder which is a hydrophilic colloid.

67. A multilayer analytical element as described in claim 66 wherein said hydrophilic colloid is gelatin.

68. A multilayer analytical element as described in claim 66 wherein said hydrophilic colloid is polyvinyl alcohol.

69. A multilayer analytical element as described in claim 37 wherein said support is composed of cellulose acetate.

70. A multilayer analytical element as described in claim 37 wherein said support is composed of poly(ethylene terephthalate).

71. A multilayer analytical element as described in claim 37 wherein said porous medium is a single blush polymer layer.

72. A multilayer analytical element as described in claim 37 wherein said porous medium also serves as a reflective background and comprises titanium dioxide dispersed in a binder.

73. A multilayer analytical element as described in claim 37 wherein said porous medium also serves as a reflective background and comprises barium sulfate dispersed in a binder.

74. A multilayer analytical element as described in claim 37 wherein said porous medium comprises diatomaceous earth dispersed in a binder.