AU616339B2

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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Australia
Prior art keywords
enzyme
substrate
zone
activity
test strip
Prior art date
1989-02-02
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AU4898590A
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Inventor
Gradimir G. Georgevich
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Abbott Laboratories

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Abbott Laboratories
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1989-02-02
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1990-02-01
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1991-10-24

1990-02-01
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Abbott Laboratories

1990-08-09
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patent/AU4898590A/en

1991-10-24
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1991-10-24
Publication of AU616339B2
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patent/AU616339B2/en

2010-02-01
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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.
ooooo i\
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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 and 3 illustrate various embodiments of the device of the present invention, the relative length of the various zones shown therein are for illustrative purposes only. In actual practice, the length of the individual zones may vary depending .upon such factors as amount and solubility of the components and the function provided by the zone. In its shortest form, a zone can be merely a line (not shown) extending across the width of the bibulous test strip 10, 30. Indicator zone 16 is preferably of sufficient length to provide a visible indication that migration has been completed. The length of the column of signal can be compared to a predetermined scale 80 having gradations 82 corresponding to enzyme or analyte concentration. The scale 80 may be incorporated (not shown) into the test strip 10, 30. Alternatively, it may be separate and form a part of a kit, The scale 80 may comprise a portion of a box or carton containing the device. -11- Zones 22 are optional accessory zones. One or more of these zones may be present and contain surfactants, buffers, enzyme cofactors, enzyme activators or other components desirable for the wicking solution. Typically the zone 22 immediately proximal to the substrate zone 18 contains buffers and other components that optimize the activity of the enzyme of interest. For example, if the enzyme is alkaline phosphatase, zone 22 should contain at least a buffer at about pH 9.4 to 10.5, preferably pH 9.8, and a phosphate Sacceptor. Wicking solution conditions, particularly pH, can be adjusted to alter the activity of the enzyme in H*9P order to increase the dynamic range. Illustrative buffers include phosphate, carbonate, barbital, diethylamine, tris, 2-amino- 2-methyl-l-propanol (AMP) and the like. Although the particular buffer employed is not usually critical, in certain instpices it may be. For example, a phosphate buffer should not be used when analyzing for acid or alkaline phosphatase activity. It is also contemplated that the accessory zones 22 could be separated from the substrate zones 18 by regions having no reactants, or placed distal to the substrate zones as taught by U.S. patent No. 4,555,484. Part or all of the device may be coated with a protectant such as fish gelatin. A. ENZYME ANALYTE The embodiment presented in Figure 1 is particularly suited to quantitating the concentration of an enzyme as an analyte of interest. Under given conditions, the activity of an enzyme is proportional to the concentration of the enzyme in the sample. Activity is determined according to the invention. Typical enzyme/analytes include alkaline phosphatase, acid phosphatase, SGOT, SGPT, and other enzymes for which a -24- 2. The method according to Claim 1 wherein said enzyme is selected from the group consisting of alkaline phosphatase, acid phosphatase, 1-galactosidase, B-D-glucosidase, horseradish peroxidase and aryl sulfatase, preferably alkaline phosphatase or acid I I C 0 C SO 0600 5* I S* S 6 h -12particularly low or high level, alone or in combination wit' other information, is clinically indicative of a disease state. In particular, Figure 2A discloses a bibulous test strip 40, like that of Figure 1, after migration of a test sample containing a high concentration of enzyme. The column of color 42 is very short and intense since the high concentration of enzyme produces substrate depletion early in the migration. As a result of high enzyme activity, the transformed substrate is immobilized more rapidly. Since a fixed, depletable quantity was present initially, the column is produced over a shorter distance during the migration and is relatively intense. In contrast, Figure 2B shows a bibulous test strip 50 after migration of a test sample containing a low concentration of enzyme. The column of color 52 is relatively long and less intense, since the low concentration of enzyme took a longer time to deplete the substrate. Thus, transformed substrate (chromophore) is immobilized over a greater distance. Quantitation of enzyme activity can be accomplished by measuring the length or intensity of the column of signal 42, 52. In the embodiment of Figure 1, the length of the column of signal (preferably color) is inversely proportional to enzyme activity and concentration. Intensity of the column of signal, however, is directly proportional to enzyme activity and concentration. B. ENZYME LABEL An embodiment of the device 30 of the present invention suitable for quantitating an analyte when the enzyme constitutes a label is presented in proximal and distal ends and incorporating a fixed, substantially depletable amount of a transformable enzyme substrate, wherein said substrate is soluble in said aqueous test sample and, upon transformation by the enzyme, becomes immobilized on the bibulous test strip -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 analyces, the conjugate usually comprises enzyme:anti-analyte antibody. However, the conjugate may also comprise avidin, biotin, lectins, complementary DNA or RNA oligonucleotides, or other analyte specific ligands complexed with an enzymatic label. The bibulous test strip 30 of Figure 3 is Sdivided into a conjugate zone 32, a capture zone 34, an accessory zone 22, a substrate zone 18, a quantitation zone 20 and an indicator zone 16. The substrate zone 18, the accessory zone 22 and the indicator zone 16 are substantially the same as those previously described, and,-for the sake of brevity, will not be discussed in detail at this point. The quantitation zone 20 is the same in that columns of signal 62, 72 are produced. The relationship of the columns 62, 72 to the analyte depends on the capture zone 34 as discussed below. In this embodiment, the conjugate zone 32 contains an excess of solulizable, analyte-specific conjugate. Methods for drying or impregnating conjugate into the zone 32 on the test strip 30 are known in the art. The conjugate is transferred to the mobile phase by dissolving therein during migration of the solvent front. By varying the content of the capture zone 34, one of ordinary skill in the art can produce a column of signal whose length is either directly or inversely proportional to the analyte concentration. Capture Ii -26transformed to a chromophoie which is insoluble in the aqueous phase, preferably wherein the transformed substrate becomes immobilized covalently by being transformed to a reactive compound which couples with a compound covalently bound to the test stri.p in the P.- t I -14- C C. C. C C. e g. C** CC C S* C zone 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 activity passing through to the substrate zone 18 is related directly (direct conjugate capture) or inversely (indirect conjugate capture) to the amount of analyte present. By the mechanism previously described, the enzyme activity quantitated by the present invention is inversely related to the length of the column of signal and directly related to the intensity of the signal column. Thus, direct capture of conjugate results in an inverse relationship between column length and analyte concentration, while indirect capture of conjugated results in a direct relationship. In a direct capture variation, the capture zone 34 contains a predetermined fixed quantity of immobilized analyte or analyte analog such as an antigen which serves to capture directly any antibody:enzyme conjugate mobilized by the solvent front which is not bound to analyte from the test sample. Only the conjugate not bound to analyte can advance with the solvent front to the quantitation zone 20. Accordingly, the greater the amount of analyte in the test sample, the greater the amount of conjugate to reach the quantitation zone. As with the first embodiment, increased amounts of enzyme produce shorter columns of color 62), and hence an inverse relationship exists between the length of the column of color and the concentration of analyte in the test sample. The length and content of the capture zone 34 contains sufficient capture reagent to capture 7 Th I i 7 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 analog in capture zone 34 so as to adjust the sensitivity of the device. For example, by placing a predetermined excess of immobilized analyte in zone 34, only those samples with analyte concentrations above a certain threshold will produce a column of color. Such an embodiment is particularly suited for screening for elevated concentrations of common serum analytes, eg. cholesterol, TSH, SGOT, SGPT, alkaline phosphatase, acid phosphatase, etc., wherein an elevated concentration is .9 associated with a particular disease state. This variation is exemplified by Weng, et al., U.S. Patent No. 4,740,468 and is well suited for any analyte for which a binding partner can be found or produced. It is particularly advantageous for detection of the following classes of analytes: the drugs of abuse, the various therapeutic and prophylactic pharmaceutical agents, hormones, microorganisms (including viruses) and antibodies to microorganisms for which antigens or antigenic determinants can be produced. Alternatively, in an indirect capture variation, the length of the column of color may be directly proportional to the concentration of analyte in the test sample by immobilizing in the capture zone 34 a second distinct ligand capable of binding the analyte. For example, the capture zone 34 can contain a second anti-analyte antibody immobilized therein, and the conjugate zone 32 can contain an excess amount of a solulizable enzyme:first antibody complex (conjugate). .i i -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 an enzyme label from the conjugate zone, the amount of enzyme passing to the substrate zone 18 is inversely related to the amount of analyte present in the sample. As previously indicated, as less enzyme label passes to the substrate zone, the transformable substrate is depleted more slowly and the column of signal is Slonger. Conversely, as more enzyme label passes to the substrate zone 18, the enzyme depletion occurs more rapidly and a shorter column of signal is produced. Accordingly, high and.low analyte concentrations in the test sample are associated with long and short columns of signal, respectively, thereby providing a direct relationship between the length of the column and the "concentration of analyte in the test sample. This method is particularly well suited for analyzing larger molecular weight antigens and macromolecules which have more than one antigenic site per molecule. Illustrative macromolecules include human chorionic gonadatropin (hCG), follicle stimulating hormone (FSH), luteinizing hormone thyroid stimulating hormone (TSH) and the like. Preferably, the conjugate zone 32 and the capture zone 34 are separated (not shown) to prevent the enzyme conjugate from becoming immediately trapped at the interface of the two zones prior to its reaction with the analyte. Also, it is preferred that zone 32 or III-IH.H.^ -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 impregnated into the conjugate zone 32. It is possible that enzyme label be selected so that it is active within the pH range of the buffer selected. For example, the enzyme label may be alkaline phosphatase, and the buffer pH range may be from 9.4 to about 9.8. Alternatively, the accessory zone 22 contains a second solulizable buffer dried or impregnated therein. The second buffer is selected to adjust the local pH to an optimum compatible with the enzyme of interest. In this manner, the pH optimum of the enzyme need not be the same as the pH optimum of the analyte-conjugate affinity reactions. METHODS OF USE In addition to the device mentioned above, the present invention further relates to a method for quantitating the activity of an enzyme. The activity quantitated, in turn, can be related to the concentration of an enzyme-analyte, or to the concentration of a non-enzyme analyte which can be complexed with an enzymatic label. The method of quantitating the activity of an enzyme employs a test strip device 10 such as that described above, and comprises the steps of: contacting the proximal end of the test strip with an aqueous test sample and allowing the test sample to migrate to the quantitation zone; and -18determining the amount of signal produced in the quantitation zone as a measure of the enzyme activity. The contacting of step includes pipetting, spotting, wicking, exposing to a fluid stream or dipping the proximal end of the test strip 10, 30 into the test sample or a solution including the test sample. This step may also serve to solubilize a reagent. For example, when a small volume of sample is pipetted onto the test strip, it may be pipetted directly onto the i. first reagent zone 22,32 to facilicate solubilization of the reagent. Preferably, after contacting the sample to the proximal end of the bibulous test strip, the test strip's proximal end 12 is contacted with a wicking solution 11 which facilitates migration. The wicking solution is preferably buffered at a pH selected to optimize the reactions in the zone, as previously described. The buffer can be added directly to the wicking solution, or can be solubilized from a reagent zone 22, 32 by the mobile phase. After the proximal end of the bibulous test strip is contacted with the aqueous 'test sample and, optionally, a wicking solution, migration of the aqueous phase is allowed to proceed toward the distal end 14 of the test strip. The next step in the method, determining the activity of the enzyme in the test sample, is accomplished either by measuring the length of the column of signal 42 or 52, or by measuring the intensity of the column of signal. The former method is preferred since it does not require elaborate instrumentation. The latter method typically does require instrumentation if more than semi-quantitative results are required. *0 O @0 0 .0. 0000 0 0 0e C 00 00*00 -19- Alternatively, by running several standards of known enzyme activity, a calibration curve is created, wherein the enzyme activity in the unknown test samples can be obtained from the calibration curve. Test samples with enzyme activities greater than the working range of the test strip are typically diluted 1:10 with 0.9% NaCI and rerun. An optional further step involves relating the enzyme activity to the concentration of the enzyme in the test sample. The second embodied device 30 may be used to determine a non-enzyme analyte which is conjugated to an enzyme label. This method comprises quantitating the activity of an enzyme label which is conjugated directly or indirectly to the analyte of interest; and relating the enzyme activity to the concentration of the analyte. The quantitating step is accomplished by measuring the length or intensity of the column of signal 62, 72 as previously described. The relating step correlates the enzyme activity to the concentration of analyte in the sample and depends on the configuration of the capture zone 34. The present invention also provides for a kit comprising any solid phase device 10, 30 embodied herein and reagents, including a wicking solution 11. A scale calibrated with gradacions 82 may optionally be incorporated in a rectangular transparent plastic housing (not shown) along with the solid phase device or as a separate component. The gradations 82 correlate the length of the column of signal to enzyme concentration and/or to analyte concentration. The following example is given by way of illustration only and should not be construed as limiting the spirit or scope of the invention as, based upon this disclosure, many variations will become obvious to those of ordinary skill in the art. EXAMPLE 1 Linear Reading System For Alkaline Phosphatase Sample Preparation Aliquots of a stock solution of alkaline phosphatase (Sigma Chemical, St. Louis, #P 6774) STRENGTH] were diluted into an aqueous solution comprising 100 mM glycine, 1 mM MgC12, 0.1 mM ZnC12 at pH 10.5 to produce solutions having approximately the following relative concentrations of alkaline phosphatase activity: 0 U/ml; 2 U/ml; 5 U/ml; 50 U/ml and 500 U/ml Strip Preparation Strips were created by adhering matrix segments together in fluid communication adjacent one another on an adhesive support in the following order to form sheets: a 65 mm wide strip of untreated Whatman #1 filter paper as part of the quantitation zone; a 5 mm wide strip of Whatman #1 filter paper impregnated with a 1 mg/ml solution of 3-indoxyl phosphate (Sigma Chemical, St Louis, #1 5505) followed by lyophilization, as the substrate zone; a 5 mm wide strip of untreated Whatman #1 filter paper as a wick. -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. Assay Protocol The proximal (wick) end of the test strip was inserted in an aqueous solution (test sample) having 500 9 U/ml of alkaline phosphatase activity. After 5 min. at room temperature, the aqueous solution (mobile phase) ascended to the distal end of the test strip. The above procedure was repeated for each of the sample alkaline phosphatase solutions prepared above. Results A blue column of color was produced by the 0 0 .oo transformed (precipitated) substrate, wherein the alkaline phosphatase activity in the test sample was inversely proportional to the length of the column of color as shown in Table I below. The reported Rf is the ratio of the measured length of color to the 70 mm length of the quantitation zone. The series of alkaline phosphatase standards which were run by the above assay method produced a calibration curve which was used to determine the alkaline phosphatase activity of subsequent unknown samples. PIP- A -22- TABLE I Sample Concentration (units/mi) 0 2 500 Length of column of color (mm) no color 38 32 18 12 Rf value n/a 54 .46 .26 .17 6* U 0 0* 00.. 0 0* 0 00 0 0 00 0090 000 000 0 0**0 0000 00 90 00 0 0 0 0 000000 Claims (12) 1. A method of quantitating the activity of an enzyme utilizing a bibulous test strip having: a proximal end and a distal end, said proximal end for receiving a test sample in an aqueous mobile phase; a substrate zone disposed between the proximal and distal ends and incorporating a fixed, substantially depletable amount of a transformable enzyme substrate, wherein said substrate is soluble in said aqueous test sample and, upon transformation by the enzyme, becomes ir .obilized on the bibulous test strip and generates a detectable 1l; and a quantitation zone disposed between i-id substrate zone and the distal end whereby, as the mobile see* S phase advances, the transformed immobilized substrate oc.« a S" produces a column of signal, the amount of signal being 0 related to the activity of said enzyme; said method comprising: contacting the proximal end of said test strip with an aqueous test sample and allowing the aqueous test sample to migrate to the quantitation zone; determining the amount of signal produced in the quantitation zone as a measure of the enzyme activity. J -24- 2. The method according to Claim 1 wherein said enzyme is selected from the group consisting of alkaline phosphatase, acid phosphatase, B-galactosidase, B-D-glucosidase, horseradish peroxidase and aryl sulfatase, preferably alkaline phosphatase or acid phosphatase and wherein said transformable enzyme substrate is 3-indoxyl phosphate. 3. The method according to Claim 1 including the additional step comprising contacting said bibulous test strip with a wicking solution. 4. The method according to Claim 1 wherein determining the amount of signal produced comprises measuring the length of the column of signal and inversely relating said length to enzyme activity, or measuring the intensity of the column of «*00 signal and relating said intensity to enzyme activity. 5. A chromatographic solid phase device for quantitating the activity of an enzyme in a sample, said device comprising a bibulous test strip having: a a a proximal end and a distal end, said proximal end for receiving a test sample in an aqueous mobile phase; a substrate zone disposed between the proximal and distal ends and incorporating a fixed, substantially depletable amount of a transformable enzyme substrate, wherein said substrate is soluble in said aqueous test sample and, upon transformation by the enzyme, becomes immobilized on the bibulous test strip and generates a detectable signal; and a quantitation zone disposed between said substrate zone and the distal end whereby, as the mobile phase advances, the transformed immobilized substrate 3 produces a column of signal, the amount of signal being related to the activity of said enzyme. 6. The device according to Claim 5 wherein said enzyme is selected from the group consisting of alkaline phosphatase, acid phosphatase, B-galactosidase, B-D-glucosidase, horseradish peroxidase and aryl sulfatase, preferably alkaline phosphrtase or acid phosphatase and wherein said transformable enzyme substrate is 3-indoxyl phosphate. 7. The device according to Claim 5 wherein the column of detectible signal comprises a column of color, the length of which is proportional to the enzyme activity. 8. The device according to Claim 5 wherein the transformed substrate becomes immobilized by being -26- transformed to a chromophore which is insoluble in the aqueous phase, preferably wherein the transformed substrate becomes immobilized covalently by being transformed to a reactive compound which couples with a compound covalently bound to the test strip in the uantitation zone. 9. The device according to Claim 5 wherein said enzyme is a label bound to an analyte of interest and said analyte of interest is reactive with en antibody or an antigen. The device according to Claim 9, wherein said test strip further comprises an capture zone disposed Gas: between said proximal end and said substrate zone for selectively passing enzyme labeled analyte to the substrate zone such that the enzyme activity quantitated in the quantitation zone is related to the amount of analyte. 69 11. A method of quantitating the activity of an enzyme utilizing a bibulous test strip substantially as hereinbefore described with reference to the Example. 12. A chromatographic solid phase device for quantitating the activity of an enzyme in a sample, said device comprising a bibulous test strip substantially as hereinbefore described with reference to the accompanying drawings. DATED this THIRTIETH day of JANUARY 1990 Abbott Laboratories Patent Attorneys for the Applicant SPRUSON FERGUSON 4 AU48985/90A 1989-02-02 1990-02-01 Method and device for quantitative chromatography Ceased AU616339B2 (en) Applications Claiming Priority (2) Application Number Priority Date Filing Date Title US30588689A 1989-02-02 1989-02-02 US305886 1989-02-02 Publications (2) Publication Number Publication Date AU4898590A AU4898590A (en) 1990-08-09 AU616339B2 true AU616339B2 (en) 1991-10-24 Family ID=23182795 Family Applications (1) Application Number Title Priority Date Filing Date AU48985/90A Ceased AU616339B2 (en) 1989-02-02 1990-02-01 Method and device for quantitative chromatography Country Status (8) Country Link EP (1) EP0381173B1 (en) JP (1) JPH02261395A (en) KR (1) KR900013307A (en) AT (1) ATE97450T1 (en) AU (1) AU616339B2 (en) CA (1) CA2009111A1 (en) DE (1) DE69004587T2 (en) ES (1) ES2048331T3 (en) Cited By (1) * Cited by examiner, † Cited by third party Publication number Priority date Publication date Assignee Title US7680421B2 (en) 2005-07-07 2010-03-16 Avago Technologies General Ip (Singapore) Pte. Ltd. Multimode optical fibre communication system Families Citing this family (14) * Cited by examiner, † Cited by third party Publication number Priority date Publication date Assignee Title DE69021327T2 (en) * 1989-11-08 1996-03-14 Chemtrak Inc Improvement in a device-free diagnostic test based on distance determination. ATE210294T1 (en) * 1995-09-08 2001-12-15 Fujirebio Kk IMMUNOASSAY DEVICE AND TEST METHOD FOR USE THEREOF DE19609838A1 (en) 1996-03-13 1997-09-18 Boehringer Mannheim Gmbh Methods and test strips for the determination of an analyte US6001658A (en) * 1996-09-13 1999-12-14 Diagnostic Chemicals Limited Test strip apparatus and method for determining presence of analyte in a fluid sample DE19651886A1 (en) * 1996-12-13 1998-06-18 Merck Patent Gmbh Means and methods for the determination of hydrolytic enzymes FR2782523B1 (en) * 1998-08-19 2000-10-06 Rhone Poulenc Nutrition Animal DEVICE FOR QUICK MEASUREMENT OF ENZYMATIC ACTIVITY ES2244310B1 (en) * 2004-02-03 2007-02-16 Universidad De Oviedo 3-INDOXYL PHOSPHATE AS SUBSTRATE OF PEROXIDASES. ES2245587B1 (en) * 2004-03-05 2007-08-01 Universidad De Oviedo Method for determining enzymatic activity, useful in clinical analysis, e.g. immunoassays and DNA hybridization, is based on detecting leuco-indigo US20050227370A1 (en) * 2004-03-08 2005-10-13 Ramel Urs A Body fluid analyte meter & cartridge system for performing combined general chemical and specific binding assays JP4980884B2 (en) * 2004-03-30 2012-07-18 ジーイー・ヘルスケア・バイオサイエンス・コーポレイション Lateral flow systems, materials, and methods GB2465814B (en) * 2008-06-19 2011-10-26 Tarig Sayed Mustafa Arbab Method,composition and device for the treatment of enzymes and saccharides disorders FI123297B (en) * 2009-05-04 2013-02-15 Valtion Teknillinen Lateral flow test strip and its manufacturing method GB201206977D0 (en) * 2012-04-20 2012-06-06 Mologic Ltd An enzyme detection device CN108949856B (en) * 2018-07-05 2021-12-28 量子高科(广东)生物有限公司 Preparation method of galacto-oligosaccharide Citations (1) * Cited by examiner, † Cited by third party Publication number Priority date Publication date Assignee Title EP0345460A2 (en) * 1988-06-09 1989-12-13 Abbott Laboratories Method and device employing covalently immobilized colored dyes Family Cites Families (2) * Cited by examiner, † Cited by third party Publication number Priority date Publication date Assignee Title US4435504A (en) * 1982-07-15 1984-03-06 Syva Company Immunochromatographic assay with support having bound "MIP" and second enzyme US4973549A (en) * 1987-06-22 1990-11-27 Chemtrak Corporation Quantitative diagnostic assay employing signal producing agent bound to support and measuring migration distance of detectable signal 1990 1990-01-31 AT AT90101859T patent/ATE97450T1/en not_active IP Right Cessation 1990-01-31 ES ES90101859T patent/ES2048331T3/en not_active Expired - Lifetime 1990-01-31 EP EP90101859A patent/EP0381173B1/en not_active Expired - Lifetime 1990-01-31 DE DE90101859T patent/DE69004587T2/en not_active Expired - Fee Related 1990-02-01 AU AU48985/90A patent/AU616339B2/en not_active Ceased 1990-02-01 CA CA002009111A patent/CA2009111A1/en not_active Abandoned 1990-02-02 KR KR1019900001227A patent/KR900013307A/en not_active Application Discontinuation 1990-02-02 JP JP2024999A patent/JPH02261395A/en active Pending Patent Citations (1) * Cited by examiner, † Cited by third party Publication number Priority date Publication date Assignee Title EP0345460A2 (en) * 1988-06-09 1989-12-13 Abbott Laboratories Method and device employing covalently immobilized colored dyes Cited By (1) * Cited by examiner, † Cited by third party Publication number Priority date Publication date Assignee Title US7680421B2 (en) 2005-07-07 2010-03-16 Avago Technologies General Ip (Singapore) Pte. Ltd. Multimode optical fibre communication system Also Published As Publication number Publication date EP0381173B1 (en) 1993-11-18 DE69004587T2 (en) 1994-05-11 EP0381173A2 (en) 1990-08-08 KR900013307A (en) 1990-09-05 ATE97450T1 (en) 1993-12-15 EP0381173A3 (en) 1991-01-09 AU4898590A (en) 1990-08-09 DE69004587D1 (en) 1993-12-23 ES2048331T3 (en) 1994-03-16 CA2009111A1 (en) 1990-08-02 JPH02261395A (en) 1990-10-24 Similar Documents Publication Publication Date Title US5200317A (en) 1993-04-06 Method and device for quantitative chromatography US5679526A (en) 1997-10-21 Threshold ligand-receptor assay US5939272A (en) 1999-08-17 Non-competitive threshold ligand-receptor assays CA1276877C (en) 1990-11-27 Multizone analytical element having detectable signal concentration zone AU616339B2 (en) 1991-10-24 Method and device for quantitative chromatography US7312028B2 (en) 2007-12-25 Highly cost-effective analytical device for performing immunoassays with ultra high sensitivity US5716778A (en) 1998-02-10 Concentrating immunochemical test device and method US4668619A (en) 1987-05-26 Multilayer homogeneous specific binding assay device US4461829A (en) 1984-07-24 Homogeneous specific binding assay element and lyophilization production method US4959307A (en) 1990-09-25 Immunoseparating strip AU600657B2 (en) 1990-08-23 Sheet-like diagnostic device JP3009155B2 (en) 2000-02-14 Immunochromatographic analysis method US4362697A (en) 1982-12-07 Homogeneous specific binding assay test device having copolymer enhancing substance US5085988A (en) 1992-02-04 Immunoseparating strip US5569589A (en) 1996-10-29 Immunoassay element and process and immunoassay US4562148A (en) 1985-12-31 Analytical element and method for preventing reagent migration EP0051213B1 (en) 1987-03-25 Homogeneous specific binding assay device, method for preparing it and analytical method using the device US5260193A (en) 1993-11-09 Immunoseparating strip US5776714A (en) 1998-07-07 Analytical, element, composition and method using modified apo-horseradish peroxidase EP0253581B1 (en) 1991-10-16 Analytical element having water-soluble polymers and determinations using same EP0517338B1 (en) 1997-03-05 Analytical element coated by a gravure process WO1991018291A1 (en) 1991-11-28 Solid support having antibodies immobilized thereon JP2576913B2 (en) 1997-01-29 Immunoassay element and immunoassay method CA2198948A1 (en) 1997-12-01 Quantitative detection of analytes on immunochromatographic strips WO1990005914A1 (en) 1990-05-31 Biological diagnostic assay system
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