AU616339B2 – Method and device for quantitative chromatography
– Google Patents
AU616339B2 – Method and device for quantitative chromatography
– Google Patents
Method and device for quantitative chromatography
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Publication number
AU616339B2
AU616339B2
AU48985/90A
AU4898590A
AU616339B2
AU 616339 B2
AU616339 B2
AU 616339B2
AU 48985/90 A
AU48985/90 A
AU 48985/90A
AU 4898590 A
AU4898590 A
AU 4898590A
AU 616339 B2
AU616339 B2
AU 616339B2
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AU
Australia
Prior art keywords
enzyme
substrate
zone
activity
test strip
Prior art date
1989-02-02
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AU48985/90A
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AU4898590A
(en
Inventor
Gradimir G. Georgevich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
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Abbott Laboratories
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1989-02-02
Filing date
1990-02-01
Publication date
1991-10-24
1990-02-01
Application filed by Abbott Laboratories
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Abbott Laboratories
1990-08-09
Publication of AU4898590A
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patent/AU4898590A/en
1991-10-24
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granted
Critical
1991-10-24
Publication of AU616339B2
publication
Critical
patent/AU616339B2/en
2010-02-01
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Status
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Classifications
G—PHYSICS
G01—MEASURING; TESTING
G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
G—PHYSICS
G01—MEASURING; TESTING
G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 – G01N31/00
G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
An improved chromatographic device and method for quantitating the activity of an enzyme utilizes a bibulous test strip as the stationary phase, an aqueous solution as the mobile phase, and a chromophore as the indicator. The enzyme activity is quantitated by transforming a substantially depletable amount of a chromogenic substrate from soluble chromogen to insoluble chromophore as the mobile phase advances, whereupon the insoluble chromophore becomes immobilized on the test strip to produce a column of color. The length and/or the intensity of the column of color are related to the activity of the enzyme. The present invention is useful for the direct determination of enzyme-analytes and for the indirect determination of analytes which can be coupled to an enzyme.
Description
1’833 S F Ref: 119814 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION
(ORIGINAL)
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FOR OFFICE USE: Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: *5*5
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Name and Address of Applicant: Abbott Laboratories One Abbott Park Road Abbott Park Illinois 60064-3500 UNITED STATES OF AMERICA Address for Service: Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, Nqw South Wales, 2000, Australia Complete Specification for the invention entitled: Method and Device for Quantitative Chromatography The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/5 L 1 iii I_4 I I
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-13- Figure 3. Typically, the enzyme is linked to a ligand or receptor capable of directly or indirectly binding the analyte of interest. The linked complex of enzyme:ligand is referred to herein as “conjugate”. A number of methods are well known to those skilled in the art for preparing the conjugate complex. For antigenic -T Th -1- METHOD AND DEVICE FOR QUANTITATIVE CHROMATOGRAPHY ABSTRACT OF THE DISCLOSURE 00 @0 0 *0 0 0 0 0
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0 0 An improved chromatographic device and method for quantitating the activity of an enzyme utilizes a bibulous test strip as the stationary phase, an aqueous solution as the mobile phase, and a chromophore as the indicator. The enzyme activity is quantitated by transforming a substantially depletable amount of a chromogenic substrate from soluble chromogen to insoluble chromophore as the mobile phase advances, whereupon the insoluble chromophore becomes immobilized on the test strip to produce a column of color. The length and/or the intensity of the column of color are related to the activity of the enzyme. The present invention is useful for the direct determination of enzyme-analytes and for the indirect determination of analytes which can be coupled to an enzyme.
i-4 -14zone 34 selectively passes conjugate to the substrate zone 18 in a manner related to the amount of analyte present. The capture zone 34 contains immobilized therein a reagent that captures conjugate directly or indirectly, so that the amount of conjugate enzyme 0 000 *0 *D 0 00 00*) 00O 00 0 o 00.
0 0 0*00 -lA- METHOD AND DEVICE FOR QUANTITATIVE CHROMATOGRAPHY BACKGROUND OF THE INVENTION The present invention relates to a method and device for quantifying the activity of an enzyme using a chromatographic test strip. More particularly, the present invention quantitates enzyme activity by transforming a substantially depletable amount of a chromogenic substrate from soluble chromogen to insoluble chromophore as the mobile phase advances, whereupon the insoluble chromophore becomes immobilized on the test strip to produce a column of color. The length and/or the intensity of the column of color are related to the activity of the enzyme. The present invention is useful for the direct determination of enzyme-analytes and for the indirect determination of analytes which can be coupled to enzymes.
The prior art contains numerous examples of.
chromatographic methods for quantitating an analyte.
See, for example, U.S. Patents 4,298,688 (Kallies); 4,059,407 (Hochstrasser); 4,168,146 (Grubb); and 4,435,504 (Zuk). All of these examples employ soluble chromogens which are converted to soluble chromophores.
Soluble chromophores present numerous problems to the development, of a quantitative chromatographic assay; namely they advance as a narrow line with the migrating solvent front, making quantitation impossible. To avoid this problem and produce a quantitative chromatographic assay, chromatographic assays were produced wherein the chromophore was developed only after migration had occurred. Although these assays avoid the migration problem, they require multi-step procedures.
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.4 substantially all the conjugate when the test sample happens to contain no analyte of interest. However, it is within the ordinary skill in the art to vary the length and amount of immobilized analyte or analyte analncr in r!’n+-iirp 7-no IA cn a, A-i;iie,- 4-1hm r 9 a.
a a 0* a 0 a a a a a a a a a. a a Another problem with soluble chromophores is that they tend to diffuse over time. Hence, the measurement of any color must be precisely timed, particularly if the distance traveled by the color is to be proportional to the quantity of analyte present. In addition, conventional reaction kinetics dictate that results be read at a precise time during development.
Copending and co-owned application Serial No. 118,148 filed November 6, 1987 discloses a method of quantitating enzyme activity employing a chromatographic strip device. A sample containing enzyme is immobilized at a reaction site on the strip and a solution of substrate/cofactors is transported over the site to form a reaction product which is transported to a detection region beyond. The rate of reactant consumption or product formation is then determined.
Others have attempted to solve soluble chromophore problems by employing dipstick tests which do not require a mobile aqueous phase. These devices have a plurality of reagents contained therein in distinct zones layers). Application of the sample to the test device causes the reagents to dissolve and mix uniformly over the dipstick. Color is uniformly distributed over the surface of the test device.
Quantitation, if possible at all, can be done only by comparison against a standard or by instrumentation capable of reflectance or transmission spectrophotometry.
SUMMARY OF THE INVENTION The present invention provides a single step method and a device for the quantitative chromatographic analysis of enzyme activity. The enzyme may be the analyte itself, or it may be a label coupled to the analyte of interest.
AS J -16- In this variation, all analyte passing through the conjugate zone 32 becomes labeled by complexing with the excess conjugate. Thereafter, enzyme labeled analyte passes to the capture zone 34 and becomes immobilized on the stationary phase by forming an antibody:analyte:conjugate sandwich with the immobilized second antibody. Because this immobilized analyte bears -3- In one aspect, the present invention is directed to an improved chromatographic solid phase device for quantitating the activity of an enzyme in a sample. The device comprises a bibulous test strip having a proximal end and a distal end, the proximal end for receiving a test sample in an aqueous mobile phase.
Disposed between the proximal and distal ends is a substrate zone incorporating a fixed, substantially depletable amount of a transformable enzyme substrate.
o The substrate must be soluble in the aqueous test sample and, upon transformation by the enzyme, become immobilized on the bibulous test strip to generate a detectable signal, such as color.
A quantitation zone is disposed between said substrate zone and the distal end whereby, as the mobile phase advances over the strip, the transformed, immobilized substrate-produces a column of signal, the amount of signal being related to the activity of said enzyme. Typically, the signal is a chromophore (preferably insoluble) so that the length or intensity of a column of color can be measured and related to the enzyme activity.
In another aspect, the invention provides a method of quantitating the activity of an enzyme, comprising the steps of contacting the proximal end of the bibulous test strip described above with an aqueous test sample and allowing the aqueous test sample to migrate to the quantitation zone, and determining the amount of signal produced there as a measure of the enzyme activity.
Finally, a method of quantitating an analyte comprises quantitating the activity of an enzyme label which is conjugated to the analyte directly or indirectly, and relating the enzyme activity to the amount of analyte.
I -17both zones 32 and 34 be buffered at a pH that maintains or optimizes the affinity between the analyte and the conjugate. Preferably, the pH ranges from about 5.5 to about 10.5, more preferably from about 6.5 to about 9.8. As in the first embodiment, buffer may be present in the wicking solution 11 or it may be dried or BRIEF DESCRIPTION OF THE FIGURES Figure 1 discloses the substrate and quantitation zones in a chromatographic device of the present invention wherein an enzyme is the analyte of interest.
Figure 2A shows the embodiment of Figure 1 in S. response to a sample containing a high concentration of enzyme.
Figure 2B shows the embodiment of Figure 1 in response to a sample containing a low concentration of enzyme.
Figure 3 discloses an embodiment of the present invention wherein the enzyme is a label bound to the analyte of interest.
Figure 4A shows the embodiment of Figure 3 in pep, response to a sample containing a high concentration of analyte.
Figure 4B shows the embodiment of Figure 3 in response to a sample containing a low concentration of analyte.
DETAILED DESCRIPTION The present invention relates to an improved chromatographic method and device for quantifying the activity of an enzyme. The method and device of the present invention employ a bibulous test strip as a solid stationary phase, an aqueous solution as a mobile phase, and a substantially depletable amount of a transformable substrate which initially is solubilized by the aqueous phase and, upon transformation, becomes immobilized and generates a signal. -18determining the amount of signal produced in the quantitation zone as a measure of the enzyme activity.
The contacting of step includes pipetting, 9potting, wicking, exposing to a fluid stream or dipping the proximal end of the test strip 10, 30 into the test Two embodiments of the device of the present invention are shown in Figures 1 and 3. The bibulous test strips 10, 30 each have a proximal end 12 and a distal end 14. The proximal end 12 is the receiving end at which the test sample is applied and from where chromatographic migration begins. The distal end 14 is where migration stops.
Optionally, the distal end 14 may contain an indicator zone 16 impregnated with a reagent which indicates when migration is complete as shown in Figures .2A, 2B, 4A and 4B by numerals 44, 54, 64 and 74, respectively. Indicator reagents which change color upon contact with the mobile phase are suitable for this zone. Dehydrated transition metal salts such as CuS3 4 or Co(NO which change color when hydrated, are suitable for aqueous mobile phases. Especially preferred are the pH indicator dyes (eg.
phenolphthalein) which can be selected to change color in response to the (buffered) mobile phase.
The “bibulous test strip” 10, 30 is any porous matrix possessing capillarity through which an aqueous solution containing the analyte may pass. Materials e* suitable for use as a bibulous test strip include various cellulose fiber containing matrices such as filter papers, chromatographic papers, ion exchange papers, cellulose acetate films, nitrocellulose films, cellulose acetate discs, cellulose thin layer chromatography discs and the like. Additional suitable materials for the bibulous test strip include starch based materials, such as Sephadex(R) brand cross-linked dextran chains, and other materials such as ceramic materials, glass fibers, films of polyvinyl chloride, and combinations of polyvinyl chloride and silica.
-6- .Especially preferred are bibulous test strips wherein the matrix material is glass fiber.
Although the thickness of the bibulous test strip may vary depending upon the size of the sample, the preferred thickness is from 0.1 to 1.0 mm. The test strip 10, 30 may be adhered to and supported by a water insoluble physical support (not shown) if desired.
The aqueous mobile phase may be the sample itself or, more preferably, a buffered wicking solution 11 containing the sample. The sample is preferably a biological fluid extracted, diluted, or concentrated from a plant or animal. Exemplary biological fluids are e’o” serum, plasma, urine, ascites fluid, peritoneal fluid, amniotic fluid, synovial fluid, cerebrospinal fluid and the like.
The buffered wicking solution comprises an aqueous solution which may contain miscible organic solvents such as lower alkyl alcohols, acetone and the like. The function of the wicking solution 11 is to transport the sample across the length of the bibulous test strip 10, 30 by capillary action. The wicking solution may contain buffers to maximize the chemical and/or immunochemical activity of tho other compolleits.
More preferably, however, buffers, cofactors, surfactants, enzyme activators and other desirable components are impregnated in an additional zone or zones (eg. 22 and 32) which are discussed in more detail below.
The substrate zone 18 contains a fixed, substantially depletable amount of a transformable enzyme substrate. It is an important aspect of the invention that the substrate be transformable. By “transformable” is meant having the ability to be i
J
-7converted by an enzyme from a substrate which is soluble in the mobile phase to a product/signal compound (transformed substrate) which is immobilized on the bibulous test strip of the solid phase. Thus, “transformation” comprehends both signal generation and immobilization. Matrices, substrates and wicking solutions are chosen so as to optimize transformation.
e.
Preferably, the signal compound generates a signal which can be detected visually. For example, it is preferred that the transformable substrate is chromogenic (a precursor), while the product 0* (transformed substrate) is a chromophore. However, fluorescent and other signals are within the scope of the present invention, provided they generate a detectible signal and become immobilized upon transformation by an enzyme.
The specific substrate chosen for the substrate zone 18 depends on the enzyme whose activity o is to be determined. One of ordinary skill in the art can easily determine a transformable substrate for a given enzyme. For example, the various alkaline and acid phosphatases can be determined with the transformable substrate 3-indoxyl phosphate (Sigma Chemical Co., St Louis #t 5505); B-D-glucosidase can be determined with the transformable substrate 3-indoxyl-B-D-glucoside (Sigma Chemical Co., St Louis); and the aryl sulfatase enzymes can be determined with the transformable substrate 3-indoxyl-sulfate (Sigma Chemical Co., St Louis #1 3875). Finally, B-galactosidase could be determined with a transformable substrate such as 3-indoxyl-B-D-galactoside.
Similarly, the fixed amount which constitutes a substantially depletable quantity also depends on the enzyme whose activity is to be determined, as well as the assay configuration. Where the enzyme is the -21- The sheets were then cut transversely into multiple strips 75 mm long by about 1.5 mm wide, with the wick defining the proximal end and the quantitation zone defining the distal end.
Alternatively, pre-cut strips can be formed by impregnating a substrate zone of desired length near the proximal end of the strip.
pT– T -r 6 0* 0* 9 0* 00 0 000 o oS 0 analyte of interest, the amount of substrate should be that quantity which will be substantially totally transformed by a given aliquot of sample having an enzyme concentration at the low end of the clinically anticipated range. Where the enzyme a label, the amount of substrate should be that .antity which will be substantially totally transformed by the minimum amount of analyte:label conjugate reaching the quantitation zone. This, in turn, is governed by the specific analyte, the sample aliquot, the capture zone configuration and other conditions. One of ordinary skill in the art can easily determine an appropriate amount of transformable substrate for a given enzyme.
Some specific examples are provided later in this specification.
Immobilization of transformed substrate on the bibulous strip encompasses both covalent immobilization and physical immobilization. In the first case, covalent immobilization can be achieved through known reactions such as linkers or derivatization of the matrix. An example of this latter approach can be found in copending U.S. application Serial Number 204,443, assigned to the same assignee as the present application and incorporated herein by reference.
Physical immobilization can be achieved through adsorption or solubility and is preferred for the present invention. A substrate is selected so that it is solubilized by migration of the aqueous mobile phase and, when transformed to the product/signal compound, is relatively insoluble in the same mobile phase. Solubilization initially releases substrate that has been impregnated or deposited, such as by drying or lyophilization, into the substrate zone 18. Typically, insolubility in aqueous phases is induced (through 1; a -22- TABLE I Sample Concentration (units/ml) 0 2 Length of column of color (mm) no color 38 32 Rf value n/a .54 .46
I-
-9transformation by the enzyme) by removal or blocking of a polar group.
The quantitation zone 20 begins where the enzymatic reaction first begins to transform the soluble transformable substrate into signal compound.
Accordingly, the zone that provides the last critical reactant to the enzymatic transformation marks the starting point of the quantitation zone 20. Since the last critical reactant typically is the solulizable transformable enzyme substrate, the quantitation zone overlaps the substrate zone 18. This overlap is evident from a side by side comparison of the drawing Figures.
The substantially depletable amount of substrate is a fixed quantity of substrate which the enzyme will transform at a rate proportional to the activity of the enzyme. Thus, the more enzyme activity m present, the faster the substrate is depleted, as shown at 42 and 62 in Figures 2A and 4A. Conversely, the less enzyme activity present, the slower the rate of S. transformation, as shown at 52 and 72 in Figures 2B and 4B. There is a fixed quantity of substrate to be depleted, so the amount of signal formed is constant.
Quantitation is achieved through the time required for the unknown enzyme activity to transform all the substrate to signal.
This time period is translated to intensity and length of the column of signal by the migration of the mobile phase. Although the wicking rate of the advancing solvent front is not absolutely constant, given a homogeneous matrix material and a constant viscosity and temperature, the rate of wicking is predictable. Depletion of the substrate tends to slow the kinetics of transformation by the enzyme. At the jj i *o -lOsame time, the wicking rate slows as the front moves toward the distal end 14, Thus, transformation of substrate (and therefore enzyme activity) is related both to the intensity of the signal and, more importantly, to the length of the column of signal produced.
This is advantageous in that the need for an 9 external timing element is eliminated. Unlike *9 o. conventional dipstick assays, the time at which the result is read is not critical. Timing is achieved by migration and solvent depletion, to make quantitation possible. Tl<5 exact mechanics of quantitation depend on the assay configuration and are discussed separately below.
Although Figures 1 3 illustrate various embodiments device present invention, relative length zones shown therein for illustrative purposes only. In actual practice, individual may vary depending .upon such factors as amount solubility components function provided by zone. its shortest form, a zone can be merely line (not shown) extending across width bibulous test strip 10, 30. Indicator 16 is preferably sufficient to provide visible indication that migration has been completed.
The column signal compared predetermined scale 80 having gradations 82 corresponding enzyme or analyte concentration.
The incorporated into Alternatively, it separate form part kit, The comprise portion box carton containing device.
-11- Zones 22 optional accessory zones. One more these contain surfactants, buffers, cofactors, activators other desirable wicking solution. Typically immediately proximal substrate 18 contains buffers optimize activity interest. For example, if alkaline phosphatase, should at least buffer about pH 9.4 10.5, 9.8, phosphate Sacceptor. Wicking solution conditions, particularly pH, adjusted alter in H*9P order increase dynamic range.
Illustrative include phosphate, carbonate, barbital, diethylamine, tris, 2-amino- 2-methyl-l-propanol (AMP) like. Although particular employed not usually critical, certain instpices be. used when analyzing acid phosphatase activity.
It also contemplated could separated from regions no reactants, placed distal taught U.S. patent No. 4,555,484.
Part all coated with protectant fish gelatin.
A. ENZYME ANALYTE embodiment presented Figure suited quantitating concentration an Under given proportional sample. Activity determined according invention. Typical>Download PDF in English
