Note: Descriptions are shown in the official language in which they were submitted.
9~.~3
-1-
ANALYTICAL ELEMENT AND METHOD FOR DETERMINATION
OF TOTAL LACTATE DEHYDROGENASE
Field of the Invention
This invention relstes to clinlcsl chem-
5 istry. In psrticular, it relstes to ~ multilsyeranalytic~l element snd ~ method for determinstion of
totsl lactste dehydrogen~se in squeous liquids~ e.g.
biologicsl fluids.
Bsck~round of the Invention
The use of di~gnostic tests in the clinical
testing of p~tients hss become incre~s$ngly common
in recent years. For example, the quantitstive
determinstion of lsctate dehydrogensse (LDH) is
extremely important in the detection of he~rt
15 disesses. Following myocardisl infsrctions, the
level of LDH in the blood rises noticesbly over it5
norm~l concentr~tion. The esrly detection of sbnor-
msl levels of LDH c~n therefore lead to a more sccu-
r~te snd rapid di~gnosi~ of`he2rt m~l~dies.
Becsuse early disgnosis of sbnorm~l hesrt
conditions is so important, 8 test for the detection
of vsriation in LDH in the blood mu~t be rspid and
simple. It also must be highly sccur~te over a
bro~d range of LDH concentrations encountered in
25 pstient testing
A significant contribution in the field of
clinical chemistry w~s the development of dry-to-
the-touch multilsyer anslytical elements which could
be used for simple, rspid snd highly Qccurate test--
30 ing of biologicsl fluids. Such elements Aredescribed, for exsmple, $n U.S~ P~tents 3,992,158
(issued November 16, 1976 to Przybylowicz et 81) and
4,258,001 (issued March 24, 1981 to Pierce et ~1).
The Pierce et a1 reference, for exsmple, describes
35 multilsyer elements comprising p~rticulste spresding
., ~
~'7~5
-2-
layers. The~e lsyers can ~l~o contain one or more
~urfsctant~ to improve layer wettability.
European Pstent Applic~tion 83/902727
(corresponding to PCT 84/00779 published March 1
5 19843 describea an assay for LDH carried out with
multilsyer snalytical element having a porou~
~preading layer containing a binder and micro-
crystalline cellulose (commercially avsilAble a~
Avicel~). However, ~uch element~ containing
lO microcrystalline cellulose ~preading l~yer~ are
difficult to manufscture on a large scsle. Further,
the a~say deacribed in this reference requires a
blank subtrsction ~tep to reduce error. Hence, 81 -
ternstive materisl~ were sou~ht for making elements
15 useful for LDH assays.
It wa~ found thst elements for LDH deter-
mination contsining the besded spresding layers
described in the Pierce et al reference noted sbove
are much essier to msnufacture on ~ large scale thsn
20 the Avicel~contain~Lng elements. It W&5 also
found that the rste curves obtained in a~says with
these element~ are more linesr than the Avicel~'
containing elements, even if no blank subtraction is
msde. However, lt wa~ ob~erved thst the ~saay of
25 LDH with the Pierce et al elements W8S unscceptably
imprec'Lse, especially at the lower levels of LDH.
Blank ~iubtrsction does not eliminate this problem.
It would be highly de~irsble to hsve an
element for LDH ass&y which i~ easily msnufactured
30 on a large ~c~le, snd which alao exhibits acceptable
precision over the entire range of L~H generslly
encountered in patient testing. It would also be
desirable to svoid blank subtraction because it com-
plicates the a~ssy and require-~ sdditionsl equipment
35 snd computer ~oftware ~n automated an~lyzers.
1 ~ ~9~ ~3
SummarY of the Invention
I have found an analytical element and
method for LDH determination that overcome the
problems noted above. In particular, the as~ay of
this invention is precise over the entire range of
LDH concentrations generally encountered ln patient
testing, and particularly at the lower concentra-
tions. Further, this improvement is achieved with-
out a blank subtraction step because the response
curves have improved linearity. The present inven-
tion is also unexpectedly less susceptible to inter-
ference by total protein and hemoglobin which may be
; in the test fluids. The improvements of this inven-
tion were achieved by the use of an analytical ele-
ment comprising a particular fluorinated surfactant
and a particulate spreading layer.
Therefore, in accordance with this inven-
tion, a dry analytical element for the determination
of total lactate dehydrogenase (LDH) comprises a
support having thereon,
a porou~ spreading layer composed of a
particulate structure comprising a plurality of
particles being bonded to each other on surface
areas of ad~acent particles where the ad~acent
particles are in closest proximity to form a
coherent, three dimensional lattice which is
essentially non-swellable in an aqueous liquid,
the element further comprising a saturated
or unsaturated fluorinated surfactant having one or
more fluorocarbon moieties, provided that when the
surfactant contains more than one such moiety, the
moieties are substantially linear.
This invention also provides a method ~or
the determination of total LDH comprising the steps
of:
~279~3
A. in the presence of a substrate for
LDH, an indicator material which provides a detect-
a~le change in re~ponse to the reaction of LDH with
a substrate for LDH, and the fluorinated surfactsnt
described above,
contacting sn analytical element with a
sample of a liquid suspected of containing LDH, the
element comprising a support having thereon ~ porous
spreading layer composed of a particulate structure
comprising a plurality of particles being bonded to
esch other on surface areas of ad~acent particles
where the ad~acent particles are in closest proxim-
ity to Eorm a coherent, three dimensional lattice
which i5 essentially non-swellable in an aqueous
liquid, and
B. detecting the rate of the detectable
change.
Brief DescriPtion of the Drawin~s
FIG. l is a graphical plot of the change in
reflection density (DR) with time for the deter-
mination of LDH using a prior art analytical element
and several calibrator test fluids as described ln
Example l below.
FIG. 2 is a graphical plot of the change in
reflection density (DR) with time for the deter-
mination of LDH using the analytical element of this
invention and several calibrator test fluids as
described in Example l below.
FIG. 3-is a bar graph showing the effect of
various surfactants on LDH assay precision using a
5 I.U./Q LDH test fluid as descri~ed in Example 2
below.
FIG. 4 is similar to FIG. 3 except that a
464 I.U./Q LDH test fluid was used as described in
Example 2 below.
~;~7~35~3
-5-
Detailed Description of the Invention
The present invention cfin be used to ad-
vsntsge tc determine the concentration of totsl lac-
tate dehydrogenase (also known as L-lactate:NAD
oxidoreductase, EC 1.1.1.27, or LDH herein) in an
aqueous liquid, such a~ a biological fluid. LDH can
be determined (i.e. qualitative, quantitative or
semi-quantitative detection~ in, for example, whole
blood, blood serum, plasma, urine, spinal fluid,
cerebral fluid, suspensions of human or animal tis-
sue, feces, saliva, sputum, and other body fluids.
Preferably, the sssay of this invention is carried
out with human whole blood or blood serum. LDH is
known to exist in five isoenzymes (i.e. LDH-l,
LDH-2, LDH-3, LDH-4 flnd LDH-5), all of which are
determined collectively with the present invention.
The unexpected advant&ges of the present
invention are achieved by the use of one or more
fluorinated surfactants in the analytical element
containing a particulate spreading layer described
in more detail below. Useful surfactant~ can be
anionic, cationic, nonionic or amphoteric. The
molecule can have more than one charged moiety and
either a net negative, positive or zero charge.
The useful surfactants are fluorinated,
which means that the molecule contains one or more
fluorocarbon moieties, each of which has one or more
hydrogen atoms replaced by fluorine atoms. The
remaining hydrogen atoms can be replaced with an-
other substituent, if desired. Each fluorocarbon
moiety generally has at least 4 carbon atoms. The
fluorinated surfactant can be saturated or unsatur-
ated. When the surfactant has two or more fluoro-
carbon moieties, these moieties are substantially
linear, meaning that they are predominantly straight
1~'79~63
--6--
chsin groups havlng no more thsn sne or two small
branche~ (e.g. methyl, fluorinsted or unfluorin-
ated). The moieties can be connected together by sn
orgsnic b~ckbone which i~ linesr or br~nched. When
5 the surfsctant hss only one fluorocarbon moiety,
thAt moiety can be branched or linear.
Represent~tive surfsct~nts are listed below~
Nonionic Surfsctsnts
Perfluoroslkylpoly(ethylene oxide) alcohols, for
10 exsmple, commercially available A~ ZONYL FSN~ from
DuPont, (Wilmington, Delswsre, U.S.A.), or as
FLUOWET OT (trade name) from American Hoechst
(Chsrlotte, North Carolin~).
Cationic Surf~ctants
Perfluoroalkyl quaternary ammonium salts, for
exsmple, commercially av~ilable as Fluorade FC 135
from 3M Corporation, (St. Paul, Minnesota, U.S.A.),
as LODYNE-106 (trade name) from Ciba-Geigy (Ardsley,
- New York, U.S.A.) or a~ ZONYL FSC~ from DuPont.
20 AmPhoteric Surfactants
Perfluoro~lkyl betaines, for ex~mple, commer-
cislly svailsble 8S SURFLON S-132 (tr~de name) from
Asshi Glass Co. (Japan), or QS ZONYL FSK from DuPont,
Fluoroalkylamino csrboxylic acid, for example,
25 commercislly ~vsilsble ss LODYNE-100 (trsde nsme)
from Cibs-Geigy, (Ardsley, New York, U.S.A.).
Anionic Surfsctsnts
Fluoroslkylsulfstes, for exsmple, FLUORTENSID
FT - 24~ from Bsyer (W. Germsny), and FLUOWET SB
30 from Americsn Hoechst and fluoroslkylslkylene-
thioalkylenec~rboxylstes, for exsmple ZONYL FSA~
from DuPont.
The Surflon~ S-132, FC-135~ FLUOWET SB
(trade name) and ZONYL FSAm m~terial~ are
35 preferred in the practice of this invention.
~1
: ~,
1.''::795~3
: --7--
The amount of surfactant used in the prac-
tice of thi3 invention will vary depending upon the
particular surfactant chosen However, generally
the surfactant is present in the element in an
amount sufficient to provide a precision in the
assay of less than a standard deviation of about
; 30 I.U./Q at LDH levels of about 5 to about
500 I.U./Q, and less than about 5% CV at LDH con-
centrations from about 500 to about 2100 I.U./~.
More preferably, the surfactant is present in an
amount sufficient to provide a precision of less
than about 11 I.U./Q at the lower LDH levels, and
less than about 3% CV at the higher LDH levels.
Aa used herein, the term precision refers
to the random error observed in the assay at various
LD~ levels. Thi~ random error can be quantified
with a term known in the art as "coefficient of
variation". Coefficient of variation (CV) is defined
a~ a ~ X times 100%, or the standard deviation "~"
about a mean X using a number of replicates.
The amount of fluorinated surfactant useful
in this invention can also be defined generally in
relation to the amount of LDH sub~trate used in the
assay. Generally, the molar ratio of surfactant to
qub~trate i~ from about 0.01:1 to about 10:l.
Preferably, the molar ratio i~ from about 0.25:1 to
about 2~5:1. Since the amount of surfactant will
vary depending upon the particular surfactant used,
the optimum molsr ratio for each surfactant-
substrate combination may vary in the entire broadrange noted above. One particular surfactant may be
especially useful st a 0.5:1 surfactant-substrate
molar ratio, whereas a second surfactant may be
especially useful st 2.0:1 ratio.
~X795~3
The fluorinated surfactant described herein
csn be incorporated into any or several layers of
the element of this invention et the t~ime of
manufacture. For example, it can be in the particu-
late spreading layer, a subbing layer, a reagentlayer, a registration layer, etc. as those layers
are described below, or in two or more of such
layers. Prefersbly, the surfsctant is in reactive
association with the substrate (described below).
This means that the surfactant and substrate are in
the same layer, or individually in layers close
enough that ~hey interact during the assay. The
surfactant and substrate can be individually incor-
porated in the element during manufacture, or added
separately or together at the time of assay. Most
preferably, the surfactant is in the particulate
spreading layer with the substrate.
LDH is determined in the prac.tice of this
invention as a result of the activity of the enzyme
on a suitable substrate to produce a detectable
change over a period of time. The detectable change
can be a signal that increases with time, e.g. an
incressing amount of potentiometric or spectrophoto-
` metric signal (such as millivolt response, fluoro-
metric or colorimetric). Alternatively, the detect-
able change can be a signal which decreases with
time such as when a detectable species is destroyed
or converted into a non-detectable species. In the
following discussions, preferred changes in fluores-
cent signals are empha~ized in the practice of thisinvention.
The analytical element of this invention
can be manufactured with incorporation of the appro-
priate reagents for the assay of LDH according to
either the forward or reverse reaction illustrated
in the following equation:
LDH
pyruvic acid + NADH < >
L-lactic acid + NAD+
9S~3
-9-
wherein NADH is nlcotinamide adenine dinucleotide,
reduced form. The forward reaGtion is preferred in
the practice of this invention. In either cas~, the
substrate is the noted acid or a suitable alkali or
ammonium salt thereof. The reagent~ can also be
added to the element ~ust prior to or during the
assay.
When incorporated into the element, the
substrate can be present anywhere in the element in
an amount which can be resdily determined by a
skilled clinical chemist. Preferably, the substrate
i9 present in the spreading layer ~described below)
in an amount of at least about 0.01, and preferably
from about 0.05 to about 2, g~m . Suitable sub-
strates are readily available comMercially from anumber of sources.
The element of this invention can also be
manufactured with incorporation of an indicator
material locsted in any suitable layer of the ele-
ment. Generally the indicator material is locatedin the registration lsyer (described below). The
indicator material is either NAD~ or NAD depend-
ing upon which of the reactions of the equation
above is used in the assay. This material i-~ pre-
sent in an amount readily known to a skilled clin-
ical chemist. Preferably, it is present in stoi-
chiometric excess. The element can contain other
reagents which can react with either NADH or NAD
in one or more enzymatic or nonenzymatic reactions
to produce a detectable signal, e.g. color dye,
fluorescence, chemiluminescence, potentiometric
change, etc. For example, NADH can be reacted with
a tetrazolium salt to produce a colored dye as
described, for example, in U.S. Patent 3,867,258
(issued February 18, 1975 to Forgione). The indi-
cator materials and other reagents which may be used
1;~ 7~ 3
-10-
in this invention can be readily obtained commer-
cially from a number of sources or prepared using
known techniques and starting materials.
The element can also contain other suitable
addenda commonly included therein to facilitate the
assay, including buffer~, surfactants (other than
the fluorinated compounds noted above), binders and
hardeners. The assay of this invention is generally
carried out at a suitable pH, e.g. from about 5 to
about 9. Therefore, it is desirable to include one
or more suitable buffers in one or more layers of
the element to maintain the des~red pH during the
assay. U~eful buffers are well known to one of
ordinary skill in the art.
The assay of this invention can be suc-
cessfully carried out with a dry analytical element
having a support and only one layer thereon. This
layer is a porous spreading layer having suitable
porosity for accomodating a test sample, diluted or
undiluted. Preferably, the spreading layer is iso-
tropically porous, which property is created by
interconnected spaces between the psrticles compris-
ing the layer. By isotropically porous is meant
that the spreading layer uniformly spreads the
applied test ~ample radially throughout the layer.
Various types of particulate matter, all
essentially non-swellable in and chemically inert
and impermeable to the li~uid components, are useful
for forming a spreading layer including, for ex-
ample, pigments (e.g. titanium dioxide, barium sul-
fate, etc.), diatomaceous earth, colloidal mate-
ri&ls, resinous or glass beads and the like. These
particulate materials can be distributed in a suit-
able binder, e.g. a colloidal or polymeric material,
as is known in the art (e.g. U.S. Patent 3,992,158,
12~795~
noted above). The amount of binder can be varied
depending upon the type of binder used and the
amount of particulate material in the spreading
layer.
Examples of other useful particulate mate-
rials which can be formed into particulate struc-
tures include the polymer particles described in
U.S. Patent 4,430,436 (issued February 7, 1984 to
Koyama et al), which particles are chemically bound
to each other through reactive groups incorporated
in the particles at the points of particle contact.
Other u~eful polymer particles are described in
Japanese Patent Publication 57(1982)-101760 (pub-
lished June 24, 1982), which particles are chemic-
ally bound to each other at points of contact with alow molecular weight adhesive compound (e.g. reac-
tion products of bisphenols, dicarboxylic acids, or
amino compounds, etc.).
Particularly useful spreading layers are
those having a particulate structure formed by
organo-polymeric particles and a polymeric adhesive
for those particles as described, for example, in U.
S. Patent 4,258,001 (noted above). Maintaining par-
ticulate integrity of the organo-polymeric particles
in the particulate structure with the polymeric ad-
hesive prevents the coalescence and flow of the
particles into the voids, snd the concentration of
adhesive at those particle surface areas of the
structure which are contiguous to ad~acent particles
insure~ that the adhesive does not flow into and
clog the voids.
Materials which can be used to prepare the
spreading layers preferred in the practice of this
invention are described in considerable detail in
the Pierce et al patent noted above. Therefore, the
. .
7 95
-12-
present disclosure is directed to 8 general descrip-
tion of the layer while noting preferred embodiments
of this invention. The thickness of the described
particulate structure can be varied depending upon
the size of the organo-polymeric particles and can
be readily determined by one of ordinary skill in
the art.
The heat-stable, organo-polymeric particles
useful in the practice of this invention are gener-
ally spherical beads having a particle size in therange of from about 1 to about 200 ~m in diam-
eter. Preferably, they have a particle size within
the range of from about 10 to about 60 ~m in diam-
eter. Particles of this size provide the appropri-
ate capillary action and test sample retention timewhich allows the desired reactions to occur.
The particles can be composed of a wide
variety of organic polymers, including both natural
and synthetic polymers, having the requisite proper-
tie~. Preferably, however, they are composed of oneor more addition polymers formed from one or more
ethylenically unsaturated polymerizable monomers,
such as addition homopolymers of single monomers or
copolymer-~ formed from two or more of such mono-
mers~ These polymers can be prepared by any of avariety of standard polymerization methods (e.g.
solution, emulsion, dispersion, suspension, etc.).
If desired, althou~h the invention is not so
limited, the particular polymer can contain one or
more reaction sites to link various interactive
compositions to the particles~
Particularly useful addition polymers are
tho~e formed by polymerizing one or more of the
following ethylenic&lly unsaturated polymerizable
monomers, the details of which are provided in the
Pierce et al patent noted above:
9S~3
(8) from 0 to 100, preferably from 0 to
about 99, weight percent of one or more amino-
substituent-free vinyl carbocyclic aromatic mono-
mers, including the tyrene monomers described in
the Pierce et al patent, as well as similar amino-
substituent-free vinyl naphthyl monomers,
(b) from 0 to about 25 weight percent of
one or more acrylic acid esters,
(c) from 0 to lO0, preferably 0 to about
75, weight percent of one or more methacrylic acid
eYters,
(d) from 0 to about 30 weight percent of
one or more ethylenically unsaturated carboxylic
acids.
(e) from 0 to about 75 weight percent of
one or more ethyienically unsaturated nitrile,
(f) from 0 to about 20 weight percent of
one or more amino-substituted vinyl carbocyclic aro-
matics, including the styrene monomers de~cribed in
the Pierce et al patent, as well as similar amino-
substituted vinyl naphthyls,
(g) from 0 to about 20, preferably 0 to
about 10, weight percent of one or more ethylenic-
ally unsaturated crosslinkable monomers, including
those which can be crosslinked with amines or gela-
tin hardeners and those having two or more ethylen-
ically unsaturated polymerizable groups,
(h) from 0 to about 20 weight percent of
one or more tertiary aminoalkyl acrylates or meth-
acrylates,
(i~ from 0 to 100, preferably 0 to about75, weight percent of one or more polymerizable,
N-heterocyclic vinyl monomers, and
(~) from 0 to about 20 weight percent of
one or more acrylamides or methacrylamides.
79~3
-14-
Particularly useful addition polymers in-
clude those listed in Table I of the Pierce et al
patent. The numbers ~n the brackets represent the
weight ratio of msnomers in the monomer blend used
5 to prepare the polymer. Poll(vinyltoluene~ t-
butylstyrene-co-methacrylic acid) L6i:37:2],
poly(styrene- _-n-butyl acrylate) [7s 25] 8nd
polystyrene are preferred polymers. The organo-
polymeric particles can contain other Hddenda, if
desired, 8S known in the art.
The polymeric adhesive which is useful in
this invention bonds the organo-polymeric particles
to one another to provide a coherent, three-
dimensional lattice in the spreading layer. The
details of particular adhesives are provided in the
Pierce et 81 patént, noted above. Generally, the
adhesive is composed of an orgsnic polymer different
from the specific polymer contained in the par-
ticles, although quite commonly the adhesive repre-
sents a polymer containing many repeating unitswhich are identical or similar to some of those
present in the polymer composition of the particles.
Prefersbly, the adhesive is composed of one
or more addition polymers formed from one or more
ethylenlcally unsaturated polymerizable monomers,
such as addition copolymers formed from two or more
of such monomers. Like the particles, the adhesive
can be prepared by any of a variety of conventional
polymerization methods.
Generally, the amount of adhesive contained
in the particulate structure is less than about lO
percent to provide optimum adhesion and liquid
spreading time, based on the weight of the particles.
Particularly useful addition polymers em-
ployed as adhesives are formed by polymerizing a
127~ ~3
-15-
blend of ethylenically unsaturated polymeriz&ble
monomers selected from the blends described as
follows, the details of which are provided in the
Pierce et al patent noted above:
A. a blend containing from about 1 to
about 35, preferably from about 10 to about 30,
weight percent of one or more amino-substituent-free
vinyl csrbocyclic aromatics as described above, and
from about 65 to about 99, preferably from about 70
to about ~0, weight percent of one or more alkyl
acrylates or methacrylates,
B. a blend containing from about 20 to
about 95, preferably from about 50 to about 95,
weight percent of one or more amino-substituent-free
vinyl carbocyclic aromatics, acrylic or methacrylic
acid esters and ethylenically unsaturated polymer-
~zable crosslinkable monomer~, and from about 5 to
about 80, preferably from about 5 to about 50,
weight percent of one or more ethylenically un-
ssturated polymerizable monomers having an activehydrogen or salts thereof,
C. a blend containing from about 15 to
100 weight percent of one or more ethylenically
unsaturated monomers selected rom the group con-
sisting of l-vinylimidazole, N-vinyl-2-pyrrolidone,
vinylbenzyl alcohol, ethyl &crylate or an acrylamlde
or methacrylamide, and from 0 to about 85 weight
percent of one or more ethylenically unsaturated
polymerizable crosslinkable monomers, and
D. a blend containlng from about 80 to
about 98, and preferably from about 85 to about 98,
weight percent of one or more acrylic or methacrylic
acid esters, and from about 2 to about 20 and pre-
ferably from about 2 to about 15, weight percent of
one or more ethylenically unsaturated polymerizable
1;~7~S63
-16-
monomers containing one or more anionic moieties
(e.g. carboxy, sulfino, sulfo, phosphono, etc. or
alksli metal or ammonium salts thereof).
Particularly useful addition polymers in-
clude those listed in Table II of the Pierce et alpatent and in U. S. Patent 4,283,491 (issued August
11, 1981 to Dappen). The numbers in the brackets
represent the weight ratio of monomers in the mono-
mer blend used to prepare the polymer. Poly(methyl
acrylate-co-2-acetoacetoxyethyl methacryl~te-co-2-
acrylamido-2-methylpropanesulfonic ~cid) [88:7:5],
poly(N-isopropylacrylamide), poly(n-butyl acrylate-
co-styrene-co-2-acrylamido-2-methylpropane sulfonic
acid, sodium salt) [70:20:10] and poly(N-vinyl-2-
pyrrolidone) are preferred adhesive polymers.
Various methods can be employed for pre-
paring the particulate structure with the above-
described particles and adhesives. Specific det~ils
of useful methods are provided in the Pierce et ~1
patent noted above.
The spreading layer of the element of this
invention is carried on a suitable support. Such a
support can be any suitable dimensionally steble,
and preferably, nonporous and transparent (i.e.
radiation trflnsmissive) material which transmits
electromagnetic radiation of a wavelength between
about 200 and about 900 nm. A support of choice for
a particular element should be compatible with the
lntended mode of detection (e.g. reflection or
transmission spectroscopy or fluorimetry). Useful
support materials include polystyrene, polyesters
[e.g. poly(ethylene terephthalate~, polycarbonates,
cellulose esters (e.g. cellulose acetate), etc.
In a preferred embodiment, the element of
this invention also comprises a second layer, some-
.~
7 95
-17-
time~ known a5 a reagent or ~ registration layer,
which contains the indicator material (e.g. NADH)
described above. Generally, the second or registra-
tion layer is immedistely ad~acent the support al-
though an optional subbing layer for adhesive pur-
poses can be interposed, if desired. The layers of
the element are generally in fluid contact with each
other, meaning that fluids and reagents and reaction
products in the fluids can pass between superposed
region5 of ad~acent layers.
The second or registration layer of the
element is preferably nonparticulate, meaning that
it is essentially free of particulate material as
compared to the spreading layer. It generally con-
tains one or more synthetic or natursl binder mste-
rials, such as gelatin (hardened or unhardened), or
other nsturally-occurring colloids, polysaccharides,
homopolymers and copolymers, such ss poly(acryl-
amide), poly(N-vinyl-2-pyrrolidone), poly(n-isopro-
pylacrylamide), poly(acrylamide-co-N-vinyl-2-pyrro-
lidone) and similar copolymers.
The element of this invention also op-
tionally contains one or more other layers including
subbing layers, radiation-blocking layers, other
5preading layer5 underneath the beaded spreading
layer described ~bove, re~gent layers, etc. as known
in the srt, e.g. as described in U.S. Patents
3,992,158 and 4,258,001, noted above.
The present invention can be performed with
any analyzer constructed to perform rate assays,
that is, to examine eech test sample more than once
over A period of time so that the rate of detectable
change can be determined.
The assay of this invention can be manual
or sutomated. In genersl, the amount of LDH in a
~X79S~3
-18-
liquid sample is determined by taking the element
from a supply roll, chip packet or other source and
physically contact~ng the spreading layer of the
element with a sample of the liquid (e.g. 1 to 200
~1). Contact of the element and sample can be ac-
complished in any suitable manner, e.g. by dipping
the element in the liquid sample or by spotting the
spreading lsyer by hand or machine with a drop of
the sample by pipette or other suitable dispensing
means. If any one or both of the substrate, indica-
tor material or fluorinated surfactant is not incor-
porated in the element during manufacture, they can
be applied to the element in a similar fashion,
separately or together.
After sample-element contact, the element
is exposed to any conditioning, such as incubation,
heating or the like, that may be desirable to
quicken ot otherwise facilitate obtaining the test
result.
The LDH determination is made by passing
the element through a suitable apparatus for detect-
ing the detectable change whether it be a potentio-
metric or spectrophotometric change. Preferably,
the determination is made by determining the rate of
disappearance of colorimetric signal (e.g. absorp-
tion) due to the reaction of NADH with pyruvic acid
to form NAD and lactic acid. The amount of LDH
can be correlated through first order kinetics to
the rate measured in the assay using standard pro-
cedures and calculations.
The following examples are provided to il-
lustrate the practice of the present invention. In
these examples, the materials used were obtained as
follows:
95~i3
-19-
Lactate dehydrogenase from Sigma Chemicsl Co.
~St Louis, Missouri, U.S.A.),
NADH from P.L. Biochemioal Co. (Milwaukee,
Wisconsin, U.S.A.),
TRITON X-100 and X-405 nonionic surfactants from
Rohm and Haas (Philadelphia, Pennsylvania, U.S.A.),
Poly(N-vinyl-2-pyrrolidone) from GAF Corp. (New
York, New York, U.S.A.),
ZONYL FSN, ZONYL FSA, ZONYL FSK, and ZONYL FSC
surfactants from DuPont (Wilmin~ton, Delaware,
U.S.A.),
MONFLUOR 31 and 51 surfactants from ICI
(Wilmington, Delaware, U.S.A.),
FC 135~ cationic surfactant from 3M Company
(St. Paul, Minnesota, U.S.A.),
FLUORTENSID FT-248 anionic surfactant from Bayer
(W. Germ~ny),
FLUOWET SB anionic surfactant from American
Hoechst (Charlotte, North Carolina, U.S.A.),
- 20 SURFLON S-132 amphoteric surfactant from Asahi
Glass Co. (Japan),
AVICEL from FMC Corp. (Phil~delphia,
Pennsylvania, U.S.A.),
LODYNE-106 and -100 cstionic and amphoteric
surfsctants, respectively, from Ciba-Geigy (Ardsley,
New York, U.S.A.), and
the remainder from Eastman Kodak Company
(Rochester, New York) or prepared using known
stsrting materials and ~ynthetic procedures.
As used in the context of this disclosure,
I.U. represents the International Unit for enzyme
activity defined as one I.U. being the amount of
enzyme activity required to catalyze the conversion
of 1 micromole of substrate per minute under stand-
ard pH and temperature conditions for the enzyme.
1;~7~S63
-20-
Example 1 - AnalYtical Element for the
Determination of Total LDH
This is a comparative example comparing sn
element of the prior art to an element of the pre-
sent invention.
An element of the prior art (see E.P.A.
83/902727 noted above) was prepared having the
following format and components:
Microcrystalline cellulose 30-80 g/m
(AVICEL)
Poly(N-vinyl-2-pyrrolidone) 0.5-2 g/m
Spreading N-tris(hydroxymethyl)- 0.05-0.2 g/m
Layer methyl-2-aminoethane
sulfonic acid (pH 7.3)
Sodium pyruvate0.05-0.2 g/m
.. . . _ _
. Gelatin (hardened)5-20 8/m
: TRITON X-405 surfactant 0.5-2.5 g/m
20 Registration N-tris(hydroxymethyl)-0.5-2 g/m
Layer methyl-2-aminoethane
sulfonic acid (pH 7.3)
NADH 0.1-0.8 g/m
.
// ~ Poly(ethylene terephthalate
An element of the present invention was
prepared having the following format and components:
9~i3
-21-
. _
Poly(vinyltoluene-_ -E~t100-200 g/m
butylstyrene-co-meth-
acrylic acid) (61:37:2
weight ratio) beads
Poly(N-isopropylacryl-0.1-2 g/m
Spreading amide)
Layer Sodium pyruvate0.01-2 gtm
N~tris(hydroxymethyl)-0.05-0.5 g/m
methyl-2-aminoethane
sulfonic acid (pH 7.3)
SURFLON S-132 fluori-0.1-10 g/m
nated surfactant
Poly(N-vinyl-2-pyrrolidone) 0.1-2 g/m
. 2
&elatin (hardened)5-20 g/m
TRITON X-100 surfactant0.05-1 g/m
Registration N-tris(hydroxymethyl)-0.5-2.5 g/m
Lsyer methyl-2-~minoethane
20sulfonic acid (pH 7.3)
NADH 0.1-0.8 g/m
. . _
/ / / / Poly(ethylene terephthalate) / / / /
/ / / Support / / /
The elcment of the prior art described
above was used to determine total LDH in several
calibrator fluids by spotting the element with a
10 ~Q sample of each fluid. The amount of LDH
(I.U./Q) was determined by measuring the change in
reflection density (DR) at 340 nm with time
(seconds) after incubation at 37~ C using a commer-
cially available spectrophotometer. No blank sub-
traction ~tep was made in generating the response
curves shown in FIG. 1. FIG. 1 shows the non-linear
curves obtained with eAch of the sample fluid A-D
(5, 1014, 1426 and 1904 I.U./Q, respec~ively).
~279~3
--22--
The ~ame tests were performed on the same
dsy on an element of the present invention described
above. No blsnk subtraction step was made in gener-
ating the response curves shown in FIG. 2. FIG. 2
shows the resulting response curves which are more
linear than those shown in FIG. 1.
Example 2 - AnalYtical Elements Containin~
Variou~ Surfactants
Several analytical elements were prepared
having the format and components of the element of
the invention shown in Example 1 except that variou~
surfactants were incorporated into the spreading
layer. The particular surfactants used are shown in
Table I below. They were present within a range of
about 0.5:1 to about 2:1 molar ratio of surfactan~
to sodium pyruvate substrate.
TABLE
Amount in
Element Surfactant Element (~/m2)
1 LODYNE-100 0.33-0.99
2 SURFLON S-132 0.36-1.4
3 FC-135~ 0.33-1.1
4 LODYNE-106 0.34-1
ZONYL FSN 0.33-o.99
25Control 1 MONFLOR 51* 0.33-1.9
Control 2 TRITON X-100** 1.1
Control 3 Hexyldecyltrimethyl- 0.18-0.53
ammonium bromide
30 *Fluorlnated surfactant having two
unsaturated and branched fluorocarbon
moietie~.
**Nonionic nonfluorinated surfactAnt.
1279S63
-23-
Each element was spotted with a 10 ~Q
aliquot of each of two test fluids containing 5 and
464 I.U./Q LDH, respectively. LDH was decermined
as described in Example 1. From the resulting data,
standard deviations, a, in I.U.t were deter-
mined for each test fluid and element. FIGS. 3 and
4 are bar graph plots of the average mefln a for
each element and the two test fluids, respectively,
across all concentrations of each surfactant used.
The acceptable a at these LDH levels, as expressed
in I.U./Q, is about 30 I.U./Q with less than
about 11 I.U./Q being the preferred a. These
~ values can be converted to % CV as described
above. It can be seen that the assays performed
with the Control elements 1-3 did not exhibit ac-
ceptable precision. Control 2 is an element pre-
pared according to U.S. Patent 4,258,001, noted
above. The asssys carried out with the elements
(1-5) prepared according to this invention exhibited
acceptable precision. These tests were made with a
blank subtraction step. The same improvement is
obtained without a blank subtraction step.
ExamPle 3 - LDH AssaY and Precision Results
A preferred element prepared according to
Exsmple 1 containing SURFL0~ S-132 surfactant in the
beaded ~preading lsyer was used to determine LDH
over a wide range of LDH concentrations (5-1904
I.U./Q). The actual precision, a (I.U./Q) for
the low LDH level test fluid determined from the
assay and the % CV for the higher LDH test fluid~,
are given in Table II below. At the low LDH concen-
trstion, it i5 known in the art to measure precision
with a since the % CV calculated at those concen-
trations would be meaningles~. For the low concen-
tration, acceptable precision is a a of about30 I.U.IQ or less. At the higher concentrations,
acceptable precision is about 5~ CV or less. These
dsta were obtained with a blank subtraction step.
7956~3
--24--
TABLE II
Test Fluid (I.U./Q) Actu~l Precision
4.6 (a)
1014 2.0~ CV
1426 2.0~ CV
1904 0.8% CV
An element similar to that descr~bed above
in this exsmple was used to determine LDH over ~n-
other range of LDH cncentrstions (203-2065 I.U./~)
lO using the same procedure except no blank subtraction
step W8S u~ed in the sss~y. Tsble III below con-
tsin~ the sctual precision of these determin~tions.
Acceptable precision W8S ~chieved in esch asssy.
TABLE III
15 Test Fluid (I.U./Q) _ctusl Precision
203 5.6 (a)
650 0.7~ CV
2065 0.7~ CV
Example 4 - LDH Ass~y Exhibitin~ Reduced
Interference
The prior srt element snd element of this
invention shown in Example 1 were evslusted for
susceptibility to interferences. E~ch element was
spottecl with 8 10 ~ sliquot ssmple of pooled
25 humsn ~lerum which hsd been treated with the poten-
tisl interferents noted in T~ble IV below. The
elements were tested sccording to the procedure
described in Example 1 snd the sdverse effect of the
interferents were determined. The devistion (~)
30 from 8 reference LDH vslue is ~ measure of the ef-
fect of the interferent. An acceptsble ~ is
+ 49 I.U./~. The results of the tests with both
elements sre ~hown in T~ble IV below.
1;~795fi3
-25-
TABLE IV
Concentration
(g/dQ) of (Q) Prior Art (~) Element
Interferent Interferent Elementof Invention
5 Totsl Protein 3.9 +25 -2
Total Protein6.4 +70 -7
Total Protein10.0 +144 -17
Hemoglobin .15 +82 -30
Hemoglobin .05 +33 -16
The data illustrate the improvement ob-
tained with the present invention using the element
containing the fluorinated surfactant in a beaded
spreading layer. The element of this invention is
less susceptible to interference by total protein
and hemoglobin,
- ExamPle 5 - Analytical Elements Contain,i~,
Various Fluorinated Surfactants
Several analytical elements were prepared
having the format and components of the element of
the invention shown in Example 1 except that various
fluorinated surfactants were incorporated into the
spreading layer at about a 1:1 molar ratio to the
substrate.
Each element was spotted with a 10 ~1
sample of each of two test fluids containing 218 and
2325 I.U./l lactate dehydrogenase, respectively.
The analyte was determined as described in Example
1, and precision data in the form of standard devia-
tion (a) or % CV were calculated. Table V below
list3 the surfactants tested and the resulting
data. All elements demonstrated acceptable preci-
sion in these tests.
1~95~j3
-26-
T A B L E V
~ with 218 I.U./l %CV with 2325
Surfactant Test Fluid I.U./l Test Fluid
ZONYL FSK 20.0 1.3
5 ZONYL FSC 14.2 0.7
FLUORTENSID FT-248 19.4 0.4
ZONYL FSA 3.8 0.6
MONFLOR 31 20.4 1.0
ExamPle 6 - Various Covera~es of_FLuorinated
Surfactant
A series of elements were prepared like the
element of the present invention shown in Example
1. The SURFLON S-132 surfactant was included in the
elements at various coating coverages. In a Control
element, the ~luorinated surfactant was omitted.
The elements weré tested with test fluids having 203
and 1918 I.U./l of lactate dehydrogenase, respec-
tively, according to the procedure described in
Example 1. The precision results, either in stand-
ard deviation (a) or ~ CV, are presented in TsbleVI below. All elements containing the surfactant
exhibited acceptable preci~ion. The Control element
exhibited unacceptable precision with the low LDH
test fluld.
T A B L E VI
Surfactanta with 203 I.U./l%CV with 1918
Level (8/m 2 Test Fluid __ I.U./l Test Fluid
O (Control) 58.3 3.3
0.037 12.0 1.9
0.187 3.5 0.9
0.37 2.4 0.7
0.74 12.4 0.9
ExamPles 7-9 - Various Element Formats
Several analytical elements of the in-
vent~on were prepared and tested as described in
2~95
-27-
Example 1 u~ing 203 and 1918 I.U./l lactate de-
hydrogenase test fluids. The elements and assays
varied from that described in Example 1 in the
following ways:
Element 7: Sodium pyruvate was placed in the
registration layer and omitted
from the spreading layer.
Element 8: Contained no sodium pyruvate. It
was added to the element st the
same time the test fluid was
applied.
Element 9: Contained no NADH. It was &dded
to the element at the same time
the test fluid W8S applied.
Table VII below shows the acceptable pre-
cision results obtsined with these test elements.
T A B L E VII
a with 203 I.U./l %CV with 1918
Element Test Fluid I.U./l Test Fluid
7 5.5 1.2
8 8.2 0.6
9 3.1 1.0
Example 10 - AnalYtical Element for the
Determination of Total LDH
This is 8 comparative example comparing an
element of the present invention contAining a fluo-
rinated surfactant having two substantially linear
fluorocarbon moieties to an element outside of the
present invention containing a fluorinated surfsc-
tant having two highly branched fluorocsrbon
moieties.
An element of the present invention was
prepared having the following format and components:
~795~3
-28-
Poly(vinyltoluene- _-E-t- 100-200 g/m
butylstyrene-co-meth-
acrylic acid) (61:37:2
weight ratio) beads 2
Poly(N-1sopropylacryl-0.1-2 g/m
Spreading amide)
Layer Sodium pyruvate0.01-2 g/m
N-tris(hydroxymethyl)-0.05-0.5 g/m
methyl-2-aminoeth~ne
sulfonic scid (pH 7.3)
FLUOWET SB fluorinated0.1-10 R/m
surfactant
Poly(N-vinyl-2-pyrrolidone) 0.1-2 g/m
---- 2
Gelatin (hardened)5-20 g/m
TRITON X-100 surfactant0.05-1 g/m
Registration N-tris(hydroxymethyl)- 0.5-2.5 g/m
Layer methyl-2-aminoethane
20sulfonic acid (pH 7.3)
NADH 0.1-0.8 g/m
/ / / / Poly(ethylene terephthalate) j / / /
/ / / Support / / /
. .
A Control element was similarly prepared
except that FLUOWET SB was replaced with 1.9 g/m
of MONFLOR 51 which is a fluorinated surfactant hav-
ing the structure:
C2F5 C~ 2F5
CF3CF2--C--CF2--CF=CFO~CH2CH2 ~ CF=CF--CF2--C--CF2CF3.
C2F5 C2F5
FLUOWET SB has the structure:
O O
ll ll
CF3~CF2~CH2CH20C--CHCH2--CO(:H2CH2~CF2~CF3 -
So3Na
1;~795~3
-29-
Both elements were used to determine total
LDH in several test fluids (containing various lev-
els of L~) by spotting the element with a 10 ~Q
sample of each fluid. The amount of LDH (I.U./Q)
was determined by messuring the change in reflection
density (DR) at 340 nm with time (seconds) after
incubation at 37 C using a standard spectrophoto-
meter. No blank subtraction step wa~ made.
From the resulting data, precision of the
respective assays was determined by calculating
standard deviations, ~, for the test fluids having
less than 500 I.U./l LDH. For the test fluids hav-
ing more than 500 I.U./l LDH, a % CV was calculated
as described above. The results are shown in Table
VIII below.
T A B L E VIII
Test Fluid Control ElementExample Element
(I.U./l LDH~ (~ or ~ CV~(a or % CV~
85.6 (~) NA
20 218 NA 4.5 (~)
464 116.2 (a) NA
697 NA 1.8~
1904 7.9% NA
2325 NA 0.6%
NA = not available
It can be ~een that the Control element did
not exhibit acceptable precision in the assay for
LDH at any LDH level it was tested at. In contrast,
the element of the present invention provided high
precision in the assay at both low and high LDH
levels. While the elements were not tested with the
same test fluids, the comparison is nonetheless
clinically acceptable because above 1900 I.U./l, the
difference between 1904 and 2325 is clinically in-
significant in measuring this analyte. The Control
~'~ 7 ~ 5~3
-30-
element was unacceptable over the entire range of
LDH concentrations. It is reasonable to believe
that the precisio~ would have been unacceptable at
218 I.U./l for the Control element since it wa~
unacceptable at both 5 and 464 I.U./l.
The invention has been described in detail
with particular reference to preferred embodiments
thereof, but it will be understood that variations
and modifications can be effected within the spirit
and scope of the invention.
