Diagnostic assay employing signal producing agent on support

Abstract: Methods and kits are provided for detecting analytes, which can be measured directly or indirectly, by production of an enzymatic product. The enzymatic product reacts with a signal producing reagent bound to a bibulous strip. Various devices are employed for producing a reaction, directly or indirectly, with the analyte and then providing for migration of the product through the bibulous strip with reaction with the reagent to produce a detectable signal. The distance from a pre-determined site to the border of the detect­ able signal is a quantitative measure of the analyte in the sample.

Description: The invention relates to diagnostic assays for analytes which result, directly or indirectly, in a substrate for an enzyme. There are many analytes present in physiological fluids which are desirably monitored in a doctor's office or in the home. Particularly, where untrained individuals are required to carry out diagnostic assays for quantitative results, the protocols and equipment should be relatively simple. The equipment should be easily manipulated and not subject to deterioration, injury, or other change which may affect the result. The protocol should have the fewest number of steps, as well as a minimal number of measurements. The fewer steps and measurements, the less likely for error to be introduced by the person performing the assay. Because of the need for individuals to measure a number of different analytes, there has been substantial attention to producing protocols and equipment which can be afforded by an individual and are relatively foolproof. In many cases, however, the assay requires electronic equipment to quantitate the result, which results in a significant expenditure by the user. Also, some of the techniques require a number of steps and measurements, which are found to introduce significant errors into the results. There is, therefore, a continuing need for alternative techniques for rapid measurement of analytes by untrained individuals. In the prior art, US-A-4 168 146; 4 298 688; 4 299 916; 4 361 537; 4 366 241; 4 435 504; 4 442 204; 4 533 629; 4 446 232; 4 447 526; and 4 454 094 are exemplary of various stick and strip assays. Sloan et al ., Clin. Chem. (1984) 30 :1705-1707; and Virapen and Harding, Clin. Chem. (1985) 31 :925, describe a stick assay for chloride. See also EP-A-0183442 for an immunoassay where the measurement region has antibodies for which the analyte competes; a dye or the like can be transported on the support. According to the invention, devices and methods are provided which include a non-diffusively bound detectable reagent uniformly bound to a bibulous support. The device is employed in conjunction with a reagent composition which includes at least one enzyme, conveniently as a mixture of enzymes, which reacts directly or indirectly with an analyte to produce a product, which product reacts, directly or indirectly, with the bound reagent to produce a detectable signal. The measured height distance from a predetermined point to a point where signal is no longer readily observed is a quantitative measure of the analyte. In the drawings: Fig. 1 is a cartoon of components of a kit embodying the subject invention. Figs. 2 a and b show an alternative embodiment employing a pad mounted on a strip in side and plan view respectively. Embodiments of the present invention provide methods and kits for detecting analytes which are, directly or indirectly, substrates of enzymes, resulting in a product which by a catalyzed or uncatalyzed reaction may react with a reagent non-diffusively bound to a support to produce a detectable signal. The method involves combining the sample with an enzyme composition, where the enzyme composition includes any additional substrates required for reaction with the analyte. Where the enzyme product does not react at a desired rate with the bound reagent, additional components may be included in the enzyme reaction mixture, on the support or in the developer solution, to enhance the rate of reaction between the enzyme product and the bound reagent. The subject invention may be employed with any analyte which can react with an enzyme or product of an enzyme to produce a product which may react, directly or indirectly, with a reagent to produce a detectable signal. The reaction with the reagent may be catalyzed or uncatalyzed, particularly enzyme catalyzed. Various analytes may be used, particularly alkanols, such as glucose, cholesterol, ethanol, etc. Other compounds which may be analyzed include urea, NAD(P)/NAD(P)H, FAD/FADH, FMN/FMNH, etc. where the cofactors may be coupled to other analytes to provide the desired result. See US-A-4,134,729 and 4,374,925 for a description of enzyme systems. For the most part the enzymes will be oxidoreductases. The concentration range of the various analytes will vary depending upon the analyte. Therefore, the concentration of the enzyme reaction components will relate to the nature of the analyte and the concentration range of the analyte of interest. The enzyme reaction composition will involve the enzyme which reacts, directly or indirectly, with the analyte and any additional cofactors or substrates necessary to produce the product which reacts with the reagent. For example, where the analyte is a cholesteryl ester, the enzyme reaction mixture will include a cholesterol esterase and cholesterol oxidase. The product of this reaction is hydrogen peroxide. Depending upon the bound reagent, the mixture may also include peroxidase, which will serve to catalyze the reaction between the hydrogen peroxide and the bound reagent, which in this case may be a leuco dye. For glucose, one may employ glucose oxidase in place of the cholesterol oxidase, while for L- or D-amino acids one may employ amino acid oxidase in place of the cholesterol oxidase. These enzymes all employ oxygen as the hydrogen recipient in the oxidation, so as to produce hydrogen peroxide. Thus, any analyte which, directly or indirectly, can be coupled with an enzyme reaction to produce hydrogen peroxide, can be employed in the subject invention. Other systems which may be employed with the same or other analytes and other enzymes include hexokinase and glucose-6-phosphate dehydrogenase with glucose and the reagent N-nitrophenylformazan, lactate dehydrogenase with lactate and the reagent methyltetrazole, alcohol dehydrogenase with ethanol and the reagent trichlorophenolindophenol, etc. See particularly, US-A-4,374,925, cols. 25-30. Various devices may be employed which involve a bibulous strip to which is non-diffusively bound a reagent capable of providing a detectable signal. For the most part, the reagents will be leuco dyes, which are stable under ambient conditions, but upon reaction with an oxidant or reductant result in production of a detectable color. While the opposite situation is possible, a colored dye which is bleached, preferably a leuco dye, will be employed as the reagent. The bibulous strip may be prepared from a wide variety of materials which allow for capillary movement of a liquid, particularly an aqueous liquid. Various materials include cellulosic materials, such as paper, nitrocellulose, silica, alumina, glass fibers, or the like. No chromatography is necessary, rather the significant factor is that the reagent be conveniently bound to the support, the support does not interfere with the production of a detectable signal, and desirably the support allows for a clear distinction between the region having the detectable signal and the region from which the detectable signal is absent. The particular material employed as the bibulous strip is not critical to this invention, but conveniently paper will be employed. The device may assume various structures. In one aspect, the device may be a simple dipstick which is calibrated, so that the region delineating between the developed detectable signal and the absence of the signal can be read from a scale printed on the device. While it will usually be desirable that the markings be affixed to the device, by the term calibrated is intended that the reagent is distributed in a predetermined manner to provide a scale related to analyte concentration. The strip may be linear, having one dimension substantially larger than the other dimension, circular, or may have a reaction pad supported by a circular or linear strip. Alternatively, a capillary may be employed filled with a bibulous material, where the filler of the capillary acts as a support for the detectable reagent. The thickness of the bibulous support will generally be from about 0.12 to 1.24 mm (5 to 50mil) and, depending upon the nature of the support, may have an inert backing. Various inert backings may be employed, such as Mylar, polyvinyl chloride, polyethylene, etc. The strips will generally be at least 1mm wide, usually at least 2mm wide, and generally at least about 2cm long, more usually at least about 3cm and not more than about 10cm long. The disks will generally be at least about 1cm in diameter, and not more than about 5cm in diameter. The capillaries will generally be at least about 0.2mm and not more than about 2mm in diameter, generally being about 2cm long, usually not more than about 10cm long. The detectable signal reagent may be bound to the bibulous support in a variety of ways. For some reagents, the reagent may be bound non-covalently, due to adsorption, with or without the aid of heat, usually not above about 60°C. Heating may take from about 5 minutes to about 6 hours. If the signal reagent is physically absorbed, the resulting colored product should also remain bound and not diffuse from its original site. Alternatively, a variety of supports may be activated, so as to provide for active functional groups, which will react with a functional group present on the reagent. The functional groups present on the reagent may be alcohols, mercaptans, amines, or the like. Various supports may be activated using cyanogen bromide, particularly supports having hydroxyl groups, spacer or linker arms may be provided, where carboxyl or amino groups may be joined to the support, functionalized alkylsilyl groups may be employed, where the functionality may be amino, carboxy, or the like, or aryl groups may be covalently bonded to the support, where the aryl groups may be functionalized with halomethyl, e.g. chloromethyl, amino, which may be diazotized if desired, or the like. These various functionalities may be joined to the reagent, either directly where the reagent has a convenient functional group, where linking does not adversely affect the properties providing the detectable signal, or a bi-functional linking group may be employed, to join the reagent to the support. The reagent may be applied to the support in a variety of ways, by itself, by itself covalently bonded to a linking group, jointly with the linking group, or the like. The concentration of the reagent in a solution applied to the support will generally be from about 0.01 to 5mg/ml, more usually from about 0.05 to 2mg/ml and will depend on the concentration range of interest of the analyte being determined. Various volatile solvents may be used such as water, methanol, ethanol, acetone, etc. and combinations thereof. The reagent is uniformly distributed on the support and may be applied to the support in a variety of ways, such as spraying, dipping, rolling, or the like. Various compounds may serve as the reagent. With hydrogen peroxide, convenient reagents include tetramethylbenzidine, 1-chloro-4-naphthol, ABTS, dicarboxidine, etc. For other enzyme products, such as ammonia reagents which may find use include bromocresol green. The enzymatic reaction mixture will include as already indicated, the appropriate enzymes and any additional cofactors, substrates, or other additives necessary to provide a reaction with the analyte and to produce a product under conditions where the product will react with the bound reagent to produce a detectable signal. The concentration of the various components of the enzymatic reaction mixture will vary widely, depending upon e.g the particular enzyme, the activity of the enzyme, the concentration range of interest of the analyte. For cholesterol, the concentration of the cholesterol esterase and cholesterol oxidase will generally be in excess and range from about 0.1 to 100 IU each, more usually from about 0.5 to 10 IU each. For glucose, the glucose oxidase concentration will generally range from about 0.1 to 100 IU, more usually from about 0.5 to 10 IU. Sufficient substrates and cofactors will be provided, so as not to be rate limiting. Generally, the concentration of the individual component will not exceed about 1 molar, usually not exceeding about 0.5 molar. Buffers will normally be present to provide a buffered solution having a pH in the range of about 6 to 10, usually about 6.5 to 9, generally being at concentrations of about 50 to 500 mmol. Various buffers may be employed, such as Tris, phosphate, carbonate, MOPS, or the like. The particular buffer will be chosen in accordance with the enzyme, so as to minimize any adverse affect of the buffer. Other additives which may be included are e.g salt to provide the desired ionic strength, stabilizers, biocides. In addition, excipients may be provided to bulk the mixture. For the most part, the mixes will be provided as dry powders, particularly lyophilized powders, following conventional lyophilization techniques. See, for example, US-A-4,447,527. Instead of having the enzymatic reaction mixture present as a powder, in some instances it may be desirable to provide for the enzymatic reaction mixture in whole, or in part, non-diffusively bound to a support. The support may be a region of the device, where the region may be in the same sheet or as a separate layer allowing for capillary transport from the layer to the remaining portion of the device. In this situation, it will usually be desirable to provide that the sample is contacted with the region containing the enzymatic reaction mixture, so that the reaction may occur at one site, followed by migration of the product through the measuring region containing the reagent providing the detectable signal. With the hydrogen peroxide producing oxidases, all that will be required is having the enzyme bound to the surface at a site outside the measuring region. The amount of enzyme bound to the surface is not critical, so long as the reaction with the analyte proceeds rapidly and substantially to completion. Various means for binding enzymes to a bibulous surface are known, many of the means employed for binding the leuco dye are available for the enzyme. Where blood is used or other medium which contains particles, such as cells, which cells may interfere with the assay, it may be desirable to separate the particles before carrying out the assay. With blood, this can be achieved using a membrane, which may be removed from the site at which the sample was placed, or be wiped clean, removing the cells or other particles from the assay device. Alternatively, the blood sample may be hemolysed, or agglutinated with reagents such as wheat germ agglutinen or anti-red blood cell antibody. In carrying out the assay, different protocols will be employed, depending upon whether the enzymatic reaction mixture is available as a separate mixture or is bound to a support in liquid transport relation to the measuring unit. The sample may be any physiological fluid, such as urine, blood, plasma, serum, or a processing fluid, or the like. Before use in the assay, the sample may be subject to various treatments, such as dilution in a buffered medium, centrifugation, extraction, concentration, chromatography, etc. The particular manner in which the sample is treated is not critical to this invention, and will depend upon the nature of the sample. As already indicated, with blood, a cell separating membrane can be provided which allows for retention of the cells and passage of the plasma through the membrane into the assay device. For further understanding of the invention, the figures will now be considered. In Figure 1, components of the kit are depicted diagrammatically. The kit would contain an alcohol swab 10 and a lancet 12, where the device is to be used for blood analysis. The patient would prick a finger or earlobe with the lancet drawing a drop of blood. A plastic syringe 14 would be provided which would draw up the blood, so as to allow the patient to measure the volume of blood drawn. A test tube 16 has a stopper 18 and the enzymatic chemical reagents 20 at the bottom. The stopper is removed and the blood may be added. Where the reagents are provided as a lyophilized powder, water may be added to dissolve the reagents prior to addition of the sample. Within limits, the amount of liquid added is not critical, since the subject invention is not concentration sensitive, but only sensitive to the absolute amount of the product which is produced. Thus, the amount of liquid to be added can be indicated roughly by an indexing mark on the test tube, not shown, so that the patient may add a sufficient amount of water to assure the dissolution of all of the enzymatic reaction components. The dipstick 22 is provided in a pouch 24 having a serrated rip corner 26 for opening the pouch to remove the dipstick 22. The dipstick 22 has a region 28 at one end for dipping into the solution, which region is not calibrated. The region also can provide for a membrane which prevents cells from passing past region 28 into the calibrated region 30. The calibrated region 30 has a number of calibration lines 32, as indicated, so as to allow for measurement of the height at which a signal can be detected. Band 34 at the end opposite region 28 and indicated with the letter D has a dye, which becomes visual upon being wetted with the eluting solution. The liquid in test tube 16 is allowed to wick up to band D and then the dipstick 22 is removed from the assay solution. One may then directly read the amount of analyte in the sample as a result of the height at which the signal can be detected. In Figure 2a and b is indicated an alternative embodiment as a side view and a plan view. The device 50 has an inert backing 52 and a bibulous strip 54, to which is bound the detectable reagent. In liquid communication with the bibulous strip 54 is pad 56. Bound to pad 56 are the enzyme reaction components, which serve to react with the analyte, either directly or indirectly, to produce a product which reacts with the signal producing reagent. On top of pad 56 is membrane 58 which serves to remove any cells or other particles from the sample, and after having served as a filtration device, may be removed from pad 56 prior to further use of device 50. As with the previous dipstick 22, a band 60 is provided to indicate when the assay may be stopped. In carrying out the assay, a kit would be provided similar to the kit previously described. However, to perform this assay, one would place the blood drop on top of membrane 58 and allow it to soak into pad 56. The membrane would then be wiped free of red blood cells and the device 50 would then be partially immersed in a solution containing the remaining members of the enzymatic reaction mixture. For example, if one were detecting cholesterol, the pad would be impregnated with cholesterol esterase and cholesterol oxidase, while the development solution would have a peroxidase in order to produce the reaction between the dye impregnated on bibulous layer 54 and the hydrogen peroxide. An inert protective barrier 62 is provided, to ensure that the developing solution passes solely through pad 56. While it is not necessary that the solution solely pass through pad 56, it is preferable that the product not be unduly diluted as it passes through the bibulous strip 54. For the most part, the developer solution will also include the various agents indicated previously for the enzyme reaction mixture, such as buffer, stabilizers, anti-foaming agents, and the like. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL Reagents Glucose, cholesterol (200mg/dl. aq. standard) 4-Cl-1-naphthol, glucose oxidase (GO), cholesterol oxidase, and horse radish peroxidase were purchased from Sigma Chemical Co. Whatman® filter paper #541 was purchased from VWR Scientific Supplies. Tris buffer was 55nM Tris, 0.1ml/l Tritron® X-100, pH 8.0 Immobilization of 4-Cl-1-naphthol On Filter Paper #541 4-Cl-1-naphthol (20mg) in 10ml ethanol was diluted with 25ml Tris buffer and the filter paper soaked in the solution with gentle agitation. After 10 min, the paper was removed, air dried and cut into 6cm x 0.4cm strips. The strips were stored in a stoppered bottle in the dark until used. Assay for Hydrogen Peroxide Standards Stock solutions of hydrogen peroxide (0.05, 0.025, 0.02, 0.005, 0.0025%) were prepared and ∼300µl aliquots transferred to disposable microcuvets. To each microcuvet was added 1 drop of 1:10 diluted HRP (∼7IU) and the solutions mixed with gentle tapping. Paper strips were dipped to a depth of about 0.25cm while maintained vertical. After wicking was complete, the strips were removed and the heights of the developed color measured. A linear curve was achieved with a sensitivity of at least 7 x 10⁻⁶M H₂O₂. Assay for Glucose One hundred mg of glucose was dissolved in 10ml Tris buffer and diluted sequentially with Tris buffer to provide six solutions ranging from 10 to 0.25mg/ml (50 to 1.4µM). Aliquots (∼240µ) were transferred to microcuvets and 30µl each of GO and HRP solutions added with gentle agitation. Individual strips were immersed in the solutions as previously described and wicking allowed to proceed until completion. The height of the blue color was then measured. A standard curve was obtained which was linear between 1.4-5.6 x 10⁻⁶M glucose. By varying the 4-Cl-1-naphthol concentration on the strip the sensitivity could be varied. Strips were prepared as previously described using different 4-Cl-1-naphthol concentrations. Standard curves were obtained using the glucose solutions described previously. The slopes obtained are as follows: It is evident from the above results, that the subject invention provides for a simple rapid protocol having few steps and a minimal number of measurements. The protocol is relatively technician independent, requiring only the measurement of the sample, all of the other aspects of the assay being substantially free of error due to user mistakes. By having an optimized amount of reagent bound to the support and employing reactions which occur rapidly and efficiently, long pathways for development of the signal are avoided, so that one obtains a relatively sharp demarcation line to provide an accurate reading. The kit is very simple, can be readily produced in a reproducible manner, and can be used in the home or doctor's office, without trained technicians necessary to perform the assay. The reading does not require expensive equipment so that it can be performed anywhere, without requiring the user to carry the equipment with them where the user may be traveling. A number of assays can be provided in a single kit, since the test tube is readily reusable, individual pouches require very little space, and reagent refills may be provided in appropriate pouches.

Claims: A method for determining an analyte which is capable of reacting, directly or indirectly, with an enzyme to produce a product which is capable of reacting, directly or indirectly, with a reagent to produce a detectable signal, employing a bibulous support comprising a calibrated zone to which said reagent is uniformly bound, said method comprising: contacting at an assay mixture receiving site a sample suspected of containing said analyte in a liquid medium, a first enzyme, any additional enzyme reaction components necessary for reaction of said analyte to produce said product, and any additional detectable signal producing agents necessary to react with said reagent to produce said detectable signal; allowing said liquid medium to migrate a sufficient distance for substantially all of said product to react with said reagent; and measuring in said calibrated zone the height of the farthest distance from said receiving site of the detectable signal as a measure of the amount of said analyte in said sample. A method according to claim 1, including the additional step of combining in a liquid medium of said sample and a reaction mixture of said enzyme, at least one said detectable signal producing agent. A method for determining an analyte which is capable of reacting with an enzyme to produce a product which is capable of reacting, directly or indirectly, with a reagent to produce a detectable signal, employing a bibulous support comprising a calibrated zone to which said reagent is uniformly bound, said method comprising: contacting at an assay mixture receiving site in a liquid medium: a sample suspected of containing said analyte, a first enzyme, any additional enzyme reaction components necessary for reaction of said analyte to produce said product, and any additional detectable signal producing agents necessary to react with said reagent to produce said detectable signal; allowing said liquid medium to migrate a sufficient distance for substantially all of said product to react with said reagent; and measuring in said calibrated zone the height of the farthest distance from said receiving site of the detectable signal as a measure of the amount of said analyte in said sample. A method according to claim 3, wherein said first enzyme is an oxidoreductase, and said product reacts with said reagent by an enzyme catalyzed reaction. A method according to claim 4, wherein the enzyme of said enzyme catalyzed reaction of said product and reagent is horse radish peroxidase. A method according to claim 4, wherein said analyte is cholesterol or precursor thereof, and said first enzyme is cholesterol oxidase. A method according to claim 4, wherein said analyte is glucose or precursor thereof, and said first enzyme is glucose oxidase. A diagnostic kit comprising a bibulous support having an assay reaction mixture site and a reagent capable of producing a detectable signal uniformly bound to said support in a calibrated measurement zone, an assay reaction mixture comprising a first enzyme capable of reacting with an analyte to produce a product capable of reacting, directly or indirectly, with said reagent, any additional components necessary for the reaction of said first enzyme, and any additional members necessary for reaction of said product with said reagent; whereby upon migration of the mixture and analyte through the bibulous support a demarcation line is produced in the calibrated zone representing as a function of its height from the site the amount of analyte in the sample. A diagnostic kit according to claim 8, wherein said first enzyme is an oxidoreductase and said additional members comprise horse radish peroxidase. A diagnostic kit comprising a bibulous support having a bibulous pad mounted on said bibulous support as an assay reaction mixture site to which a first enzyme is bound, said first enzyme being characterized by being capable of reacting with an analyte to produce a product, and a reagent capable of producing a detectable signal uniformly bound to said support in a calibrated measurement zone, said product and said reagent being capable of reacting directly or indirectly to produce a detectable signal; and an assay reaction mixture comprising any additional compounds necessary for the reaction of said first enzyme, and any additional members necessary for reaction of said product with said reagent; whereby upon migration of the mixture and analyte through the bibulous support a demarcation line is produced in the calibrated zone representing as a function of its height from the site the amount of analyte in the sample.

Inventors: KHANNA PYARE, SINGH PRITHIPAL, KHANNA, PYARE, SINGH, PRITHIPAL
Applicants: PYARE KHANNA, SINGH PRITHIPAL, KHANNA, PYARE, SINGH, PRITHIPAL
Application year: 1988
Main classification: G01N33
IPC codes: A61B5, C12Q1, G01N33