CA1279563C - Analytical element and method for determination of total lactate dehydrogenase - Google Patents
Analytical element and method for determination of total lactate dehydrogenaseInfo
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- CA1279563C CA1279563C CA000508457A CA508457A CA1279563C CA 1279563 C CA1279563 C CA 1279563C CA 000508457 A CA000508457 A CA 000508457A CA 508457 A CA508457 A CA 508457A CA 1279563 C CA1279563 C CA 1279563C
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Abstract
ANALYTICAL ELEMENT AND METHOD FOR DETERMINATION
OF TOTAL LACTATE DEHYDROGENASE
Abstract of the Disclosure A multilayer analytical element exhibits improved precision in the assay of total lactate dehydrogenase (LDH). The element also provides reduced susceptibility to total protein and hemo-globin interferences in the assay. The element contains a porous spreading layer composed of a particulate structure. A fluorinated surfactant is also used in the assay. This surfactant has one or more fluorocarbon moieties provided, that when the surfactant has more than one such moiety, they are substantially linear. The surfactant can be incor-porated in the element or added at the time of the assay.
OF TOTAL LACTATE DEHYDROGENASE
Abstract of the Disclosure A multilayer analytical element exhibits improved precision in the assay of total lactate dehydrogenase (LDH). The element also provides reduced susceptibility to total protein and hemo-globin interferences in the assay. The element contains a porous spreading layer composed of a particulate structure. A fluorinated surfactant is also used in the assay. This surfactant has one or more fluorocarbon moieties provided, that when the surfactant has more than one such moiety, they are substantially linear. The surfactant can be incor-porated in the element or added at the time of the assay.
Description
9~.~3 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 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 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 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 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 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 . .
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 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 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 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-.~
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 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 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 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 . _ 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 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.
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 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 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 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 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 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 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.
FLUOWET SB has the structure:
O O
ll ll CF3~CF2~CH2CH20C--CHCH2--CO(:H2CH2~CF2~CF3 -So3Na 1;~795~3 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 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.
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 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 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 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 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 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 . .
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 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 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 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-.~
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 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 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 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 . _ 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 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.
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 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 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 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 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 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 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.
FLUOWET SB has the structure:
O O
ll ll CF3~CF2~CH2CH20C--CHCH2--CO(:H2CH2~CF2~CF3 -So3Na 1;~795~3 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 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.
Claims (19)
1. A dry analytical element for the determination of total lactate dehydrogenase (LDH) comprising a support having thereon a porous spreading layer composed of a particulate structure comprising a plurality of particles being bonded to each other on surface areas of adjacent particles where said adjacent particles are in closest proximity to form a coherent, three dimensional lattice which is essentially non-swellable in an aqueous liquid, said element further comprising a saturated or unsaturated fluorinated surfactant having one or more fluorocarbon moieties, provided that when said surfactant contains more than one such moiety, said moieties are substantially linear.
2. The element of Claim 1 further com-prising a substrate for LDH wherein the molar ratio of said fluorinated surfactant to said substrate is from about 0.01:1 to about 10:1.
3. The element of Claim 2 wherein the molar ratio of said fluorinated surfactant to said substrate is from about 0.25:1 to about 2.5:1.
4. The element of Claim 1 further com-prising a second layer containing an indicator mate-rial which provides a detectable spectrophotometric change in response to the reaction of LDH with said substrate.
5. The element of Claim 1 wherein said fluorinated surfactant is located in said spreading layer.
6. The element of Claim 1 wherein said surfactant has only one fluorocarbon moiety.
7. A dry multilayer analytical element for the determination of total lactate dehydrogenase (LDH), said element comprising a support having thereon, in order and in fluid contact, a registration layer comprising an indica-tor material which provides a detectable spectro-photometric change in response to the reaction of LDH with a substrate for LDH, and an isotropically porous spreading layer comprising said LDH substrate and composed of a particulate structure comprising:
(i) a plurality of heat-stable, organo-polymeric particles non-swellable in and impermeable to an aqueous liquid, and having a particle size of from about 1 to about 200 µm, and (ii) an adhesive, in an amount of up to about 10 weight percent of said particles, com-prising an organic polymer different from that of said particles, substantially all of said adhesive being concentrated on surface areas of adjacent par-ticles where said adjacent particles are in closest proximity, and bonding said particles into a coher-ent, three-dimensional lattice which is essentially non-swellable in said liquid, said element containing a saturated or unsaturated fluorinated surfactant containing one or more fluorocarbon moieties, provided that when said surfactant contains more than one such moiety, said moieties are substantially linear, which surfactant is present in a molar ratio to said substrate of from about 0.01:1 to about 10:1.
(i) a plurality of heat-stable, organo-polymeric particles non-swellable in and impermeable to an aqueous liquid, and having a particle size of from about 1 to about 200 µm, and (ii) an adhesive, in an amount of up to about 10 weight percent of said particles, com-prising an organic polymer different from that of said particles, substantially all of said adhesive being concentrated on surface areas of adjacent par-ticles where said adjacent particles are in closest proximity, and bonding said particles into a coher-ent, three-dimensional lattice which is essentially non-swellable in said liquid, said element containing a saturated or unsaturated fluorinated surfactant containing one or more fluorocarbon moieties, provided that when said surfactant contains more than one such moiety, said moieties are substantially linear, which surfactant is present in a molar ratio to said substrate of from about 0.01:1 to about 10:1.
8. The element of Claim 7 wherein said fluorinated surfactant is selected from the group consisting of a perfluoroalkyl quaternary ammonium salt or a perfluoroalkyl betaine.
9. The element of Claim 7 wherein said fluorinated surfactant is located in said spreading layer.
10. The element of Claim 7 wherein said particles comprise an addition polymer formed from one or more of the following ethylenically unsatur-ated polymerizable monomers:
(a) up to 100 weight percent of an amino-substituent-free vinyl carbocyclic aromatic, (b) up to about 25 weight percent of an acrylic acid ester, (c) up to 100 percent of 8 methacrylic acid ester, (d) up to about 30 weight percent of an ethylenically unsaturated carboxylic acid (e) up to about 75 weight percent of an ethylenically unsaturated nitrile, (f) up to about 20 weight percent of an amino-substituted vinyl carbocyclic aromatic, (g) up to about 20 weight percent of an ethylenically unsaturated crosslinkable monomer, (h) up to about 20 weight percent of a tertiary aminoalkyl acrylate or methacrylate, (i) up to 100 weight percent of an N-heterocyclic vinyl monomer, and (j) up to about 20 weight percent of an acrylamide or methacrylamide, and said adhesive comprises an addi-tion polymer formed from ethylenically unsaturated polymerizable monomers selected from the following group:
A. a blend containing from about 1 to about 35 weight percent of one or more amino-substituent-free vinyl carbocyclic aromatics and from about 65 to about 99 weight percent of one or more alkyl acrylates or methacrylates, B. a blend containing from about 20 to about 95 weight percent of one or more amino-substituent-free vinyl carbocyclic aromatics, acrylic or methacrylic acid esters and ethylenically unsaturated polymerizable crosslinkable monomers, and from about 5 to about 80 weight percent of one or more ethylenically unsaturated polymerizable monomers having an active hydrogen or salts thereof, C. a blend containing from about 15 to 100 weight percent of one or more ethylenically unsaturated monomers selected from the group con-sisting of 1-vinylimidazole, N-vinyl-2-pyrrolidone, vinylbenzyl alcohol, ethyl acrylate or an acrylamide or methacrylamide, and up to 85 weight percent of one or more ethylenically unsaturated polymerizable crosslinkable monomers, and D. a blend containing from about 80 to about 98 weight percent of one or more acrylic or methacrylic acid esters, and from about 2 to about 20 weight percent of one or more ethylenically un-saturated polymerizable monomers containing one or more anionic moieties.
(a) up to 100 weight percent of an amino-substituent-free vinyl carbocyclic aromatic, (b) up to about 25 weight percent of an acrylic acid ester, (c) up to 100 percent of 8 methacrylic acid ester, (d) up to about 30 weight percent of an ethylenically unsaturated carboxylic acid (e) up to about 75 weight percent of an ethylenically unsaturated nitrile, (f) up to about 20 weight percent of an amino-substituted vinyl carbocyclic aromatic, (g) up to about 20 weight percent of an ethylenically unsaturated crosslinkable monomer, (h) up to about 20 weight percent of a tertiary aminoalkyl acrylate or methacrylate, (i) up to 100 weight percent of an N-heterocyclic vinyl monomer, and (j) up to about 20 weight percent of an acrylamide or methacrylamide, and said adhesive comprises an addi-tion polymer formed from ethylenically unsaturated polymerizable monomers selected from the following group:
A. a blend containing from about 1 to about 35 weight percent of one or more amino-substituent-free vinyl carbocyclic aromatics and from about 65 to about 99 weight percent of one or more alkyl acrylates or methacrylates, B. a blend containing from about 20 to about 95 weight percent of one or more amino-substituent-free vinyl carbocyclic aromatics, acrylic or methacrylic acid esters and ethylenically unsaturated polymerizable crosslinkable monomers, and from about 5 to about 80 weight percent of one or more ethylenically unsaturated polymerizable monomers having an active hydrogen or salts thereof, C. a blend containing from about 15 to 100 weight percent of one or more ethylenically unsaturated monomers selected from the group con-sisting of 1-vinylimidazole, N-vinyl-2-pyrrolidone, vinylbenzyl alcohol, ethyl acrylate or an acrylamide or methacrylamide, and up to 85 weight percent of one or more ethylenically unsaturated polymerizable crosslinkable monomers, and D. a blend containing from about 80 to about 98 weight percent of one or more acrylic or methacrylic acid esters, and from about 2 to about 20 weight percent of one or more ethylenically un-saturated polymerizable monomers containing one or more anionic moieties.
11. The element of Claim 7 wherein said substrate is pyruvic acid or a salt thereof, and said indicator materlal is nicotinamide adenine dinucleotide, reduced form.
12. A method for the determination of total lactate dehydrogenase (LDH) comprising the steps of:
A. in the presence of a substrate for LDH, an indicator material which provides a detect-able change in response to the reaction of LDH with a substrate for LDH, and a saturated or unsaturated fluorinated surfactant having one or more fluoro-carbon moieties, contacting an analytical element with a sample of a liquid suspected of containing LDH, said element comprising a support having thereon a porous spreading layer composed of a particulate structure comprising a plurality of particles being bonded to each other on surface areas of adjacent particles where the adjacent particles are in closest proxim-ity to form a coherent, three dimensional lattice which is essentially non-swellable in an aqueous liquid, and B. detecting the rate of said detectable change, provided that when said fluorinated sur-factant contains more than one fluorocarbon moiety, said moieties are substantially linear.
A. in the presence of a substrate for LDH, an indicator material which provides a detect-able change in response to the reaction of LDH with a substrate for LDH, and a saturated or unsaturated fluorinated surfactant having one or more fluoro-carbon moieties, contacting an analytical element with a sample of a liquid suspected of containing LDH, said element comprising a support having thereon a porous spreading layer composed of a particulate structure comprising a plurality of particles being bonded to each other on surface areas of adjacent particles where the adjacent particles are in closest proxim-ity to form a coherent, three dimensional lattice which is essentially non-swellable in an aqueous liquid, and B. detecting the rate of said detectable change, provided that when said fluorinated sur-factant contains more than one fluorocarbon moiety, said moieties are substantially linear.
13. A method for the determination of total lactate dehydrogenable (LDH) comprising the steps of:
A. in the presence of a substrate for LDH
and an indicator material which provides a detect-able change in response to the reaction of LDH with said substrate, contacting the analytical element of claim 1 with a sample of a liquid suspected of con-taining LDH, and B. detecting the rate of said detectable change.
A. in the presence of a substrate for LDH
and an indicator material which provides a detect-able change in response to the reaction of LDH with said substrate, contacting the analytical element of claim 1 with a sample of a liquid suspected of con-taining LDH, and B. detecting the rate of said detectable change.
14. The method of Claim 13 wherein said detectable change is a spectrophotometric change.
15. The method of claim 14 wherein said detectable change is a colorimetric change.
16. The method of Claim 15 wherein the rate of disappearance of absorption is detected.
17. A method for the determination of total lactate dehydrogenase (LDH) comprising the steps of:
A. contacting the analytical element of Claim 7 with a sample of a liquid suspected of containing LDH to provide a detectable colorimetric change, and B. detecting the rate of said colorimetric change.
A. contacting the analytical element of Claim 7 with a sample of a liquid suspected of containing LDH to provide a detectable colorimetric change, and B. detecting the rate of said colorimetric change.
18. The method of Claim 17 wherein said liquid is human serum or whole blood.
19. The method of Claim 17 wherein the rate of disappearance of absorption is detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000508457A CA1279563C (en) | 1985-06-03 | 1986-05-06 | Analytical element and method for determination of total lactate dehydrogenase |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US740,163 | 1985-06-03 | ||
| US848,613 | 1986-04-07 | ||
| CA000508457A CA1279563C (en) | 1985-06-03 | 1986-05-06 | Analytical element and method for determination of total lactate dehydrogenase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1279563C true CA1279563C (en) | 1991-01-29 |
Family
ID=4133066
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000508457A Expired - Lifetime CA1279563C (en) | 1985-06-03 | 1986-05-06 | Analytical element and method for determination of total lactate dehydrogenase |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1279563C (en) |
-
1986
- 1986-05-06 CA CA000508457A patent/CA1279563C/en not_active Expired - Lifetime
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