Note: Descriptions are shown in the official language in which they were submitted.
lOSGZ82
, .;
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.
- 2 -
- .
1056Z8Z
,
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.
,
'
-- 3 --
:; ` 1056Z8Z
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.
. .
Description of the_Drawings
Figures 1 - 5 depict alternative configurations of pre-
... .
ferred embodiments of the analytical web of the present invention.
-4-
t - - `- -~
'
`' : ~ '. '
., ,~ ' ,
.j
~. 1056;~8Z
Figure 6 shows graphic results of analyses performed
with the webs of the present invention.
.~
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.
, 1056Z8Z
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
-:
--6--
~,: :. . .
~ 105628Z
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.
--7--
" 1(~56Z8Z
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
-8-
. : , :
:
.
s~; `i
~ 1056Z8Z
~- 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_
':
1056Z8Z
; 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.
;
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
--10--
, . ~ : -:
; ` 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
~ ?
` 1056Z~Z
"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
-12-
'
-
:
~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
-14-
. .
` ` 1056~,82
'` "
;~ 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
..~,
1056Z8Z
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.
.: :
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
-16-
;.~,!,
'.'''.' " '
`` 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
-18-
. : ~ - :,
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
'`:
--19--
. .:
. .
-`` lOS6Z8Z
.
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
- 20 -
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:
;
-21-
.
~0562flZ
.
D
, ~
.. .~ O
~ ~C .
~ .~ ~r
~ U~
' ~ $ ~
.: ~ U~
., ~ ~
D ~
a) u~ a)
: ~ ~ ~~ O
.~ a) O X
~1
oa~
O S~ ~ ~ .
.' ~ O
~c a) ~ o
+ ~CS) ~ .
~, I 1 -o
,~ x
o
Q~ ~ ~o
J~
~o
a)
o~
h X ~
U~ \\ :
11 \o
O O ~C " '
U S
3 ~:
-22-
1056Z8Z
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
- 23 -
:
` 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
- 24 -
~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
-25-
, . .
1056Z82
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
-26-
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;
-27-
:.: ' -' ... .
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~
1056Z8Z
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
- 29 -
- - . . :
:: '
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.
-30-
1056Z8Z
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
-31-
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
-32-
1056Z82
.
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).
-34-
1056Z82
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.
-35-
- ..
