JPH02130469A - Dry chemical reagent release product, manufacture thereof and analysis and test kit using the same - Google Patents

Abstract

PURPOSE: To use an object for the clinical assay and immunoassay of biological fluid by forming it using a film that is subjected to conditioning treatment containing the total amount of a reagent that is specikal to the reaction with one or more types of specimens to be concerned. CONSTITUTION: As a method for manufacturing a reagent discharge object, for example, a conditioning agent is combined with one or two or more types of interaction substances of a reagent system and at the same time are absorbed by a film. In a produced film 3, a sample acceptance surface 2 of the film 3 essentially does not transmit cell and fine particle substances. On the other hand, an opposite surface 4 is relatively porous and the natural gap rate of a matrix 6 of the film 3 extremely decreases from its initial value by absorbing a conditioning agent, an indicator, an interacting reagent, and a flow adjuster. As a result, the relative density of the initial film increases effectively. Therefore, the object is effective to analyze a biological fluid containing, for example, cells and fine particle substances.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to certain articles and analytical methods using the same. In particular, the present invention is concerned with providing highly effective dry chemical reagent release articles and incorporating the reagent release articles into a plurality of different test strips. The term "dry chemical reagents" is intended to include reagent systems used in both clinical chemistry assays and immunoassays. This dry chemical reagent release article is useful for rapid and effective analysis of biological fluids. One of the unique features of this article is that samples containing cellular and particulate matter or macromolecules can be analyzed without prior separation of such endogenous materials from the sample. Such separation is generally desirable and essential in some assays to prevent such substances from interfering with the detection of reporter molecules that indicate the presence of the analyte of interest.

Research into simple and effective means of performing analytical tests on heterogeneous fluid samples, particularly biological samples, has spanned over 25 years. Much of the early work in this field was directed toward developing reagent formats that were simple yet compatible with effective analytical protocols. One of the relatively simple types of such reagent-releasing articles is a "dry chemical reagent system."
, i.e. by providing a reagent system that is absorbed into an absorption medium, dried and reconstituted simply by addition of the fluid sample/absorption of the fluid sample by the absorption medium.

The patent and technical literature relating to the development and improvement of such dry reagent release articles is numerous (known). Included are so-called "paper" or "absorbent" layered articles. An increasingly popular format of dry reagent release articles is based on single- and multi-layer absorbent media.

One of the early and relatively successful applications of absorbent media formats for dry chemical reagent release articles was the development of a series of assays to analyze whole blood. Some of the early patents indulging in this field included heterogeneous fluids (whole blood)
The application of this format to the analysis of glucose and other common analytes of interest (i.e., urea, cholesterol) is disclosed. The following list includes patents for simple analytical devices (test strips) that illustrate the application of dry chemical reagent systems to the analysis of heterogeneous biological fluids: U.S. Pat. No. 3,061.523.
No. 3.552°925 (Mr. Free)
No. 3.607 (Mr. Fetter);
゜093 (Mr. Stone (3tone)); Same No. 3.
No. 092.465 (Mr. Adam X)
3.298.789 (Mr. Mast); and 3.630.957 (Rev
)Mr).

US Pat. No. 3,061,523 (Free) discloses a standard basic chemical reagent system for the colorimetric determination of glucose in biological samples. The chemical reagent system disclosed by Mr. Free is ``dip 5
tick) for use in connection with the J test. In a representative example of Free's invention, a face (ie, a stick or test strip) is pretreated with Free's novel chemical composition. This in-phase reagent treatment portion can then be contacted with a sample suspected of containing glucose. The intensity of the color developed as a result of such contact is compared to a control or standard to provide a semi-quantitative determination of glucose in the sample.

In a particular example of Free's device, a dry chemical reagent release article is produced by first dissolving the reactive components in a gelatin base and then impregnating strips of filter paper with this dry chemical reagent system.

This is accomplished simply by soaking the filter paper in the gelatin base/reagent system for a period sufficient to impregnate the reagents into the filter paper. The filter paper thus treated is then dried. The gelatin component of the impregnating solution is reportedly essential for the uniformity of the color development. Presumably, the presence of gelatin controls or inhibits fluid movement within the filter paper to minimize chromatographic separation of reagents and/or samples.

The portion of the filter paper containing the dry chemical reagent system is then deposited with -drops of blood (preferably whole blood) and allowed to react with the reagents contained in the filter paper for approximately 60 seconds, after which time the blood (preferably whole blood) is removed from the filter paper. Wash and remove red blood cells. The intensity of the color indicator that develops as a result of the interaction of the glucose with the reagents in the filter paper is then observed or measured. Regarding Free's reagent system, the recommendation (but not a requirement) to wash away red blood cells from the surface of the test strip indicates that removal of red blood cells is desirable but not essential for observing/measuring colored indicators. means. Washing away red blood cells from the test strip would introduce analytical errors into the above-described analysis, impairing the accuracy of the test results.

If the technician performing this test is handling specimens from patients that may contain infectious microorganisms or viruses, the requirement to wash away red blood cells from the specimen may expose the technician to unnecessary exposure to potential infection. resulting in exposure.

U.S. Patent No. 3,552,925 (Mr. Fetzter)
1 illustrates improvements to the glucose test element disclosed in the Free patent. Mr. Fetter analyzes a whole blood sample by dividing it into its serum component and its red blood cell component (erythrocytes and other color-forming (colo)
Disclosed are methods and devices for effectively separating the components (rformirrg). Mr. Fetter is
He achieved this separation by treating a limited area of the sample collection device on his test member with some water-soluble salt. Red blood cells (and other colored components of whole blood) are removed by contact of the whole blood sample with water-soluble salts in the test material.
A localized reaction between the blood and water-soluble salts occurs, resulting in the separation of serum components from the whole blood sample. The serum fraction is therefore free to move or diffuse through the test element. Normally, movement and/or diffusion of serum components occurs by capillary action or some other passive transport mechanism. Depending on the sample application method and the nature of the test medium, the serum is effectively transported and distributed to other limited chemically reactive areas of the test element containing test reagents. The test reagents of the test element are specific for one or more analytes of interest in the serum fraction (ie, glucose, galactose, urea, uric acid, phenylaniline and/or various enzymes).

Various examples of Mr. Fetter's test members include individual separated (monolayers with chemically treated zones (Figures 1 and 8)).
, or multilayer structures (Figures 3-7) where one chemical treatment is limited to each layer of the laminate.

Fetter also shows that the same matrix can be used to hold both separation reagents and reagents specific to the analyte of interest. In this latter example of Fetter's invention, a whole blood sample is deposited on one side of the strip, which is held in a horizontal position. After the sample has properly penetrated into the matrix containing both separation and test reagents, the test strip is inverted and the desired color development occurs at the site opposite to the whole blood sample application site. Observe. It is unclear whether there was any concern that colored blood components may interfere with the color development and/or observation of indicator species. However, it is clear that at low analyte concentrations, highly colored blood components can interfere with and/or obscure the presence of the indicator from being visible/detectable to the naked eye.

Canadian Patent No. 659.059 is one of the early attempts to produce a single dry chemical reagent release article in a monolayer format. The dry chemical reagent release article of this patent is
The article is based on producing a common slurry containing an absorbent media component (i.e. cellulose acetate) along with the reactive component.

The resulting slurry is then cast and dried. The reactive components of this article are physically entrapped within the resulting film/coating. The physical properties of this film/coating help absorb fluid samples without releasing (leaching) reactive components.

No. 3,092,465 (Adams) discloses a diagnostic device for detecting glucose in a heterogeneous biological fluid sample (ie, blood or urine). The device consists of an absorbent medium containing a dry chemical reagent system specific for reaction with glucose, indicating reaction with glucose by producing a macroscopic color change. This absorbent medium is provided with a "protective coating" in the form of a semi-permeable membrane. This membrane is suitable for relatively large molecules (
i.e., proteins and hemoglobin), but
It is selective in that it allows free passage of the fluid fraction containing glucose to be absorbed by the absorbent medium. Because of this protective coating, the test strip supporting the dry chemical reagent release article can be immersed or submerged in the test fluid without absorbing the colored components of the sample. The protective coating on the sample receiving surface can then be wiped free of interfering particles or colored substances before monitoring the color change. This operation of wiping the surface of the analytical member may introduce analytical errors into the above-mentioned analysis, thereby impairing the accuracy of the test results. Similar errors can be expected when removing such deposits by cleaning or other physical means.

U.S. Pat. No. 3,298,789 (Mast) discloses a test article for detecting glucose in a heterogeneous fluid sample (ie, whole blood).

The test article consists of a layer of absorbent material impregnated with a dry chemical reagent system. This layer is coated with a smooth semi-transparent film of transparent ethylcellulose. This test device detects glucose in whole blood by simply depositing a whole blood sample (2-3 drops) on the surface of a semi-permeable film coating applied to a material impregnated with reagents. used to. After a short period of time, the fluid fraction of the sample is absorbed by the absorbent material. The cellular (colored) fraction of the sample is then wiped off the surface of the semi-permeable film so that the indicator produced by the reaction of glucose with the reagent in the absorbent layer can be observed/measured. Therefore, the surface properties of this semi-transparent film are important for operating this device in an analytical atmosphere.

In particular, the smoothness (surface porosity) of this protective film is important and needs to be small enough to prevent cellular components and hemoglobin fractions from penetrating into the film surface. As noted in the discussion of the Free and Adams patent, the operation of removing cells and colored deposits from the sample-receiving surface of the test member can introduce analytical errors into the above-described analysis.

U.S. Pat. No. 3,607,093 (Stone) discloses a liquid semipermeable membrane having a uniform chemical composition;
Discloses containing a dry chemical reagent system within an IJ box. The semipermeable membrane that Stone selected for use in this device is similar to Mast's protective film in its surface properties.

The analytical protocol using Stone's device is also similar to Mast's device, allowing the removal of cellular debris and colored substances from the sample and the observation/measurement of reaction products that indicate the presence of the analyte of interest. The sample-receiving surface must be physically wiped in order to Stone's analytical method suffers from similar drawbacks mentioned above common to those of Free, Mast and Adams, namely the removal of cellular debris and colored deposits from the material-receiving surface of the analytical element, which is necessary before monitoring the indicator color phenomenon. There is a disadvantage that analysis errors may be introduced by physically removing or washing.

Further modifications and improvements have been made to the basic dry chemical reagent format described above. These changes and improvements:
providing multiple test areas on a common test member;
The focus is on increasing the accuracy of the correlation between indicator color development and analyte concentration; and controlling to a greater extent the absorption/distribution of the sample within the absorbent medium in which the reagent is impregnated. Next patent: U.S. Patent No. 3.847.82
No. 2 (Mr. Shuey); No. 3.802
．． No. 842 (Mr. Lange); No. 3.96
No. 4.871 (HOChStra
SSer); and No. 4.160.008 (Fenochetti) are examples of improvements and changes to dry chemical reagent formats.

No. 3,802,842 (Lange et al.) discloses a test strip incorporating a dry chemical reagent system in which the sample receiving surface of an indicator (reagent) layer is covered with a fine mesh. The indicator layer can be supported on a "colorless" and "transparent" support. Attaching a fine mesh to the test member reportedly improves the distribution rate and uniformity of the sample over the surface of the indicator layer. This uniformity of distribution also reportedly significantly improves the reproducibility of results.

US Pat. No. 3,847,822 (Xuei) discloses what is termed an "asymmetric" membrane containing a polymer blend of polyvinylpyrrolidone and cellulose acetate. Although this membrane composition reportedly has improved transport of solutes in the blood, particularly insulin, it is substantially impermeable to albumin.

U.S. Pat. No. 3,964,871 (Hochstrassel) discloses a disposable indicator (test strip) with an integrated color density scale that directly relates analyte concentration to test sample.

One can reportedly simply dip the indicator into the test sample and observe the development of the color phenomenon in various areas of the device. Thus, the color development of the indicator in a particular area of the test strip can be directly related to a particular concentration of analyte.

U.S. Pat. No. 4,160,008 (Fenocheti) discloses a test device for use in clinical analysis of samples on a common support for a variety of analytes.

The inventors elevated the reagent-specific layer above the common support and attached absorbent paper to the support to allow one analytical site to be run-off by another analytical site.
Each different analytical test is effectively separated and performed on a separate area on a common support, ensuring that there is no cross-contamination.

The next generation of developed dry chemical reagent systems (at least in terms of complexity) are multilayer members having at least three separate active layers. These separate working layers are the diffusion layer, the reagent layer and the signal (indicator) layer.

The use of multilayer films to quantify glucose in whole blood has been the subject of many publications and patents. The following list shows technical publications in this field:
Walter, B. Nidry Reagent Chemistry in Clinical Analysis, Analytical Chemistry, Vol. 55, No. 4, No. 498-51.
Page 4 (April 1983); Kaam, Henry Shi-6
et al.: Multilayer Film Elements for Clinical Analysis: General Concepts “Clinical Chemistry” Volume 24, No. 8, No. 1335
~1342 pages (August 1978); Spade, Richard W., et al.: Multilayer Film Elements for Clinical Analysis: Applications to Representative Chemical Determinations [ Clinical Chemistry) IJI, Vol. 24, No. 8, pp. 1343-1350 (1978,
August); Okubo, Akuki et al.: Plasma Glucose Concentrations of the Whole, Brad, As Determined with a Multilayer Film Analytical Elements "Clinical Chemistry" Volume 27, No. 7, No. 1287-12
90 pages (July 1981); Okubo, Akuki et al.: Multilayer Film Analysis for Urea.
Nitrogen in Blood, Shearam, Orr Plasma, Clinical Chemistry, Vol. 30, No. 7, pp. 1222-1225 (July 1984); and Lovechock, Patricia et al.: Dry Reasient.
Strips of Nest for Determination by Theophrin in Shearum, Clinical Chemistry, Volume 31, No. 5.

pp. 737-740 (May 1985). The following list shows the corresponding patents in this field: U.S. Pat.
No. 042.335 (Mr. Clement)
4, No. 059.405 (Sodickson (Sod)
1ckson et al.); No. 4.144.306 (
Mr. Figueras); No. 4.25
No. 8.001 (Pierce); and No. 4.366, 241 (Rom et al.).

U.S. Patent No. 4.042.335 (Mr. Clement)
A series of multilayer analytical elements suitable for performing chemical analysis of whole blood samples is disclosed. All of Clement's methods deposit the test sample onto the reagent layer either directly or from a diffusion layer. The reagent layer contains the complete amount of chemicals needed to react with a particular analyte that may be present in the test sample.
ENT). If a specimen is present, it is classified as “detectable 5pec”.
ies) J is generated or released from the reagent layer, which is called "registration".
on) layer. By registration layer is meant a layer whose sole function is to provide a medium or repository in which something detectable can be observed or measured. To avoid interference (obscuring) in observing or measuring the detectable, the registration layer is free of both the test sample and the reagents used to generate the diffusible. In the preferred embodiment of Clement's test member, an optical shield (an "emission blocking layer") is also provided between the reagent layer and the registration layer.

This optical shield effectively optically isolates the detectable from other components that may interfere with its detection and/or measurement.

As is clear from the above description, Mr. Clement attempted to separate the individual functions of his analysis elements into separate layers.

Although this technique is extremely attractive to manufacturers with the ability to produce multilayer components, it is unduly complex by its very nature and extremely difficult due to the mechanical instability of the composite component. accompanied by. Particularly if this test member is intended to be used by an individual in a self-testing environment, this composite member must be supported on additional members to provide physical integrity to the multilayer member and avoid unintended bending. It is necessary to prevent potential separation of the various layers contained therein.

US Pat. No. 4,059,405 (Sodickson et al.) discloses a method and apparatus for analyzing glucose in whole blood samples. Sodickson's method uses polyox (p
The reaction sites are first created by pre-forming indentations in filter paper treated with (olyox) resin. Diametrical ICl11
Reaction sites are physically formed in the treated filter paper by pressing a confining ring onto the treated filter paper. A glucose reagent is then deposited on the reaction site formed by the ring, and the reaction site is dried. An ultrafiltration membrane is placed over this well and a piece of paper with a spot of dried blood is placed in adjacent relationship with the ultrafiltration membrane. The dried blood spot is then restored by adding saline. The equipment (i.e. press) used in Sodickson's method is
The reconstituted blood sample remains confined within the open well for a short mixing period. During this warm-up period, the soluble components in the whole blood sample are redissolved in saline, passed through an ultrafiltration membrane, and contacted with the glucose test reagent in the polyox-treated filter paper.

Since the cellular components of the blood remain on the ultrafiltration membrane, they do not interfere with the color development and/or measurement of the glucose indicator.

The method disclosed by Sodicksson is cumbersome (requires reconstitution of the blood sample and requires relatively complex equipment to separate cellular components from whole blood samples), as seen in Example 1d. ), is not useful for self-testing purposes.

U.S. Pat. No. 4,144,306 (Figuras) discloses a multilayer analytical member similar to the Clement patent mentioned above. Figulus' chemistry is such that the interaction of the analyte with the non-diffusible reagent in the reagent layer releases "pre-formed detectables" that migrate from the reagent layer into the registration layer. It differs from Mr. Clement's in that it allows for This pre-generated detectable is then observed or measured in the registration layer. Mr. Figiniras,
As described in this Example 6, Figulus' method is applied to whole blood glucose analysis. The separation of colored and cellular components of whole blood is accomplished essentially the same way in the Figginius patent as in the Clement patent. When a whole blood sample is introduced into the reagent layer of Figginius' member, the pre-formed diffusible photographic dye (ph)
1c dye) is released, and this dye is then free to migrate into the registration layer. The Figginius patent states that to avoid obscuring or interfering effects in detecting the dye from non-diffusible color components in the reagent layer (i.e., sample and reagent), the reagent layer and the registration layer must be An optical shield of the same type (radiation blocking layer) must be present between them. The limitations and shortcomings noted in the discussion of Clement's patent also apply to Figginius' multilayer analytical member. However, the Figinilas patent adds the additional complexity of effectively immobilizing the reagent within the reagent layer and storing the preformed indicator until released by the analyte of interest.

The mechanical properties of the composite member are important because of the need to maintain fluid contact between the various components in the Figuras composite member. Therefore, the Figulus multilayer member requires a (transparent) support layer to provide physical integrity to the device, as previously discussed for the Figulus patent.

U.S. Pat. No. 4,258,001 (Pierce et al.) discloses a multilayer analytical member (of the type described in the Clement and Figuras patents mentioned above) that incorporates a unique diffusion layer. are doing. The diffusion layer in Pierce's patent is described as being essentially a "non-fibrous material." In one preferred example described by Pierce (Figure 3), the diffusion layer can contain an "interactive composition" (test reagent) for reaction with the analyte in the test sample. Pierce also uses the device to analyze whole blood, serum and urine. Whole blood can be applied directly to the Pierce member. Even in the presence of red blood cells, it has been reported that spectroscopic Photometric analysis is not disturbed (column 26).

(See lines 49-61).

As is clear from the above-mentioned patents, Pierce's device is designed to "take up" whole blood samples. Both cellular and non-cellular components of whole blood are absorbed by the diffusion layer. Therefore, the diffusion layer of Pierce's patent was not intended as a means for separating the cellular fraction of blood from the serum fraction.
It's not what is expected either. When clinical assays for enzyme-based diagnostics are applied in a diffusion layer (as is the case in glucose analysis), low glucose concentrations may be There is a risk that clinically meaningful results may be distorted by obscuring the results.

Transport and diffusion of biological samples is relevant in dry reagent systems for performing clinical assays as well as in dry reagent systems used for immunoassaying the same biological fluids. The following U.S. patents: U.S. Pat. No. 4.049.647 (Deutsch) and U.S. Pat.
No. 6, 214 (Tom et al.) is an example of literature related to immunoassays in this field.

U.S. Pat. No. 4,094,647 (Deutsch) discloses a linear wick with a limited area for placement of reagents and samples. The end of the core is placed in a vertical position in a developer fluid, and the fluid is drawn up into the core by capillary action.

As the fluid is drawn up into the core, it transports the reagent and sample from their respective locations into contact with each other. One or more of these reagents can be immobilized on the core, thus transporting reagents and samples to the immobilized reagent, and unreacted or mobile substances from the immobilized reagent. A developer fluid can be used. The site with the fixed reagent can then be observed or measured for the presence of the analyte.

US Pat. No. 4,366,241 (Tom et al.) discloses a device for non-chromatographic immunoassay of biological fluids. In Tom's device, a test member with a relatively small test area is coated with an immobilized binding agent ("mip" or "member of a 1 mmu-nolo immunological binding pair").
logical binding pair (called J). The test area of the device is the only point of entry for the biological sample and is used to receive and remove the biological sample by depositing it directly or by immersing it in the test fluid. . When an analyte is included in the biological sample, the analyte is selectively immunochemically bound to an immobilized binding agent specific for this analyte in the test area. The remaining components of the sample, including its fluid components, are drawn from the test area to a second member in adjacent fluid contact with the test area.

The function of the second member is to wick or draw the biological sample through the test area and into the test member.

Therefore, sample components that do not bind in the test zone are drawn into the test member and away from the test zone.

In immunological test elements of the type disclosed by Tom, pretreatment of the test area effectively confines the specimen and test reagents (i.e., labeled indicators) to the analysis site, so that the reagents and sample The problem of migration, which is common in phase systems used for clinical chemistry analysis, is essentially eliminated. However, the immunoassay format in Tom's patent is not without drawbacks, the most common being nonspecific binding of interfering substances in the reaction zone and the difficulties that can be associated with detecting low levels of analyte. be.

US Pat. No. 4,466,232 (Liotta) discloses a multilayer test device for immunoassays. This device and test protocol are quite similar to the Figulus patent mentioned above. Addition of a sample containing the analyte of interest causes movement of what was previously immobile. In the scheme disclosed by Liotta, the mobile component of the reagent system conjures part of an immunological binding pair.
gate) (hereinafter referred to as "conjugate J"). This conjugate is carried into the stack, where it can produce something detectable.

As can be seen from the above discussion of literature suitable for whole blood analysis, each type of test element generally requires multiple layers in its preferred configuration. In the literature in which monolayer (component) test devices are taught, with the exception of one publication (U.S. Pat. The potential chemical and optical interference of red blood cell populations (and other colored components of blood) in the detection of blood is not recognized or appreciated. When using immobilization techniques (such as those described in the Fetter, Deutsch, and Thom patents mentioned above), under certain conditions the specificity of the binding reaction may become non-discriminatory. In the case of the Fetter patent, attempts to remove red blood cells and other colored components from the sample by means of certain salts (which are absorbed into the test medium) were not made without limitation.

In Fetter's monolayer member, the serum fraction is radially separated from the colored components of the blood, so that the analyte is distributed over a relatively large area. Analytes can easily escape detection if they are present in low concentrations. Therefore, some magnification mechanism (ie, the use of oxygen in conjunction with a chromogenic substrate) may be required to make low levels of analyte visible to the naked eye.

Immunochemical devices and techniques of the type described in the Deutsche, Thom and Liotta patents mentioned above are not compatible with whole blood analysis. Although test members can be washed to remove cellular debris (as suggested in the Free patent), the effect of such additional steps on the immunochemical binding process is unknown. This is expected to introduce analytical error into such analyses.

Such handling of the test element would also be expected to interfere with the detection of reactions revealing the analyte or with (partial) migration of the indicator indicative of the analyte of interest. Furthermore, during such physical removal procedures of cellular and colored debris, clinicians and test personnel are necessarily exposed to potential infection by these components of the blood being removed from the test material. It will be done.

In short, it is clear that the prior art lacks a simple yet accurate device for whole blood analysis. Although such devices have been proposed, they are complex and cannot be self-tested unless fixation or additional support is provided.
seff-testing) and generally lack the sensitivity to allow differentiation of different levels of analytes over a wide clinical concentration range. Moreover, all such devices mentioned above (with the exception of Mr. Stone's patent)
They have one common drawback. That is, if red blood cells are not physically removed from the test element or an optical shield (blocking layer) is not provided between the colored component of the sample and the resulting indicator compound of the clinical assay, the observation / It is not possible to optically (and in some cases chemically) isolate red blood cells and other colored components of whole blood from the measurement location.

The object of the invention is to remedy the above-mentioned and related drawbacks of the prior art.

In particular, a primary object of the present invention is to provide a dry chemical reagent release article for use in clinical and immunoassays of biological fluids.

Another object of the invention is that cells, particulate matter and macromolecules (
A dry chemical reagent release article that is effective for the analysis of biological fluids containing substances (hereinafter collectively referred to as interfering substances) and that does not require pretreatment (i.e., filtration or centrifugation) or dilution of the biological fluid. Our goal is to provide the following.

Another object of the invention is a dry chemical reagent release article suitable for the rapid and effective analysis of whole blood for one or more analytes of interest without the need for prior separation of serum from cell fractions. Our goal is to provide the following.

Another object of the present invention is to quickly and effectively analyze urine for one or more analytes of interest without the need for prior concentration and/or chemical extraction of the analytes of interest from the urine sample. An object of the present invention is to provide a dry chemical reagent release article suitable for use in a dry chemical reagent release article.

Another object of the invention is to provide a dry chemical reagent release article suitable for rapidly and effectively analyzing saliva for one or more analytes of interest.

Another object of the invention is to provide a dry chemical reagent release article that is self-contained and does not require additional equipment or extensive training to perform diagnostic assays or interpret results.

Another object of the present invention is to provide a dry chemical reagent release article that is stable and retains potency until placed in use by contact with biological fluids.

The foregoing and related objects provide a dry chemical reagent release article comprising a conditioned membrane containing a total amount of reagent that is specific for reaction with one or more analytes of interest. This is achieved by Interaction of the analyte with such a reagent is clearly indicated by the release or production of an indicator molecule that indicates the presence or absence of the analyte in the sample. The sample-receiving surface of the membrane is relatively dense and is therefore impermeable to cells, particles, and macromolecules that could potentially interfere with the reaction of the analyte with the reagent system and/or the detection of the Balk molecule.

The opposite surface of the membrane, on the other hand, has a significantly lower density (higher porosity), allowing for the injection of reagent systems during fabrication and the generation of reporter molecules representing the analyte of interest, allowing for diffusion. can be made visible to the naked eye.

The dry chemical reagent release article of the present invention can be formed as a component of a test strip used to analyze whole blood samples, urine or saliva. In any of these configurations, the sample receiving surface of the membrane is the exclusive means of accessing the biological fluid to the dry chemical reagent system.

The invention will now be explained by way of example with reference to the drawings.

The dry chemical reagent release articles of the present invention include (a) a conditioned membrane in which a fluid sample is analyzed; (b) analyte-specific reagent distribution within the membrane; and (c) is unique in its ability to effectively separate different fractions of a heterogeneous sample. This separation separates sample fractions from those that could potentially interfere with the reaction of the analyte with reagents specific for its detection, or that would interfere with the detection of reporter molecules that indicate the presence or absence of the analyte in question. It is essential for effectively separating the components of the sample being analyzed. The ability to physically prevent a portion of the sample from being absorbed by the membrane depends on a number of factors, including the inherent density of the sample-receiving surface of the membrane and the distribution of components in the reagent-releasing article within the matrix of the conditioned membrane. Based on combination.

Membranes that are suitable as reservoirs for dry chemical reagent systems are anisotropic in that, prior to conditioning, there is a density gradient from one flat surface to the other. This density gradient is primarily created as an accessory to the manufacturer. In particular, when the membrane components are poured from a slurry, slurry particles tend to settle on the support surface before or during evaporation of the poured fluid. Once the applied fluid has evaporated, the membrane forms a self-supporting film that can be separated from the supporting surface. Sedimentation of the slurry particles tends to produce a denser film adjacent the support surface. Components from which membranes can be made include both synthetic and natural materials: nylon, cellulose acetate, cellulose acetate-nitrate esters, and mixtures thereof.

Of course, the physical properties of the membrane (tensile strength, thickness, etc.) must be matched to the fabrication of the test strips. That is, the membrane must have sufficient dimensional stability and integrity to be able to sequentially absorb and dry conditioning agents, reagent cocktail rings, and/or indicators without losing its physical strength. Preferably, the physical attributes of the membrane also provide sufficient durability and flexibility for use in automated methods of continuously manufacturing test strips. Additionally, the physical properties of the membrane must otherwise be compatible with the absorption and retention of aqueous fluids in the environment in which it is intended to be used.

The membrane also needs to be relatively chemically inert, ie, essentially inert to both the components of the chemical reagent system within the membrane and the components of the sample with which it is reacted. However, it must be forewarned that certain inherent properties of the membrane and/or IJ hooks may exhibit some degree of affinity for components of the reagent system and/or components of the fluid sample. This natural suction force can, in some cases, immobilize the sample cocktail and/or sample components onto a portion of the membrane, causing one of the reagent cocktails/sample components to
It can be advantageously used to achieve species separation or anisotropic distribution. This type of separation based on the natural binding affinity of the membrane can be used advantageously in both clinical chemistry and immunoassays.

It is also necessary that the optical properties of the membrane allow for effective observation and monitoring of the indicator that indicates the reaction. This requirement is intended to provide a background of sufficient contrast to allow observation of the indicator at relatively low concentrations. If the indicator has a fluorophore, the background fluorescence of the membrane should be minimal or essentially non-fluorescent at the monitored wavelength of interest.

If the inherent properties of the membrane are not conducive to effective monitoring of the indicator, it may be desirable to incorporate pigments into the dry chemical reagent system. For example, some membranes potentially suitable for use in the present invention are colored or transparent. The introduction of pigments into the chemical reagent system provides a suitable background during indicator measurements.

However, membrane transparency is limited by turbidimetry and photo-fluorometric assays in which the presence or absence of analyte in a sample is indicated as a change in turbidity in the fluid phase.
It can be used advantageously in both cases (assay). Typical examples of dry chemical reagents for such turbidimetrically measured reaction systems include pigments that change color, transparency, or background, which are absorbed into the membrane, preferably together with conditioning agents. .

1. Manufacture of Glucose Test Strips To further illustrate the unique characteristics described above, the dry chemical reagent release articles of the present invention are described for test strips containing reagents specific for the detection of glucose in whole blood. do.

Standard chemical reagents for glucose detection are described in the above-mentioned US Pat. No. 3,061,523. The basic complement of interactive substances necessary in the reagent to produce a color reaction for glucose detection are glucose oxidase, peroxidase, 0-tolidine dihydrochloride, and buffering salts/acids.

To illustrate this, the fluid-absorbing media used in this reagent release article include relatively dense surfaces (pore size >0.5 microns) and relatively sparse surfaces (pore size > Various synthetic membranes (1.0 micron) with
Preferably a cast membrane
Any of the above can be used.

The order in which the components of the dry chemical reagent system are adsorbed onto the membrane is generally determined by considerations such as chemical compatibility and/or other factors related to solubility in a common solvent.

Since the O-tolidine solution is highly acidic, it is incompatible with the enzymes of the reagent system, thus allowing each to be absorbed separately onto the membrane.

First, protein, glucose, dextrin or dextran, starch, polyethylene glycol (PEG),
Polyvinylpyrrolidone (PVP).

Conditioning the membrane by treatment with a first solution containing the same or equivalent. The purpose of such conditioning treatment is twofold.

(a) to effectively reduce voids within the membrane matrix; and (b) to aid or accelerate the absorption of fluid fractions of the biological sample. In another example of a method of making this reagent release article, the conditioning agent can be combined with one or more interactants of the reagent system and simultaneously absorbed into the membrane. When a conditioning agent is combined with an interacting agent in a reagent composition, it necessarily causes absorption of the indicator molecules prior to absorption by the membrane of the conditioning agent.

If such a membrane conditioning process is performed independently of the interactants of the reagent system, the membrane is dried under controlled conditions and then exposed to the indicator molecule or a chemical precursor of the indicator molecule. and a second solution containing the second solution. Absorption of the indicator molecule or its precursor can occur from either the relatively sparse or relatively dense surface of the membrane, but is preferably from the sparse surface.

After absorbing the above solution, the membrane is again dried under controlled conditions to ensure that this solution does not escape.
ive) Maintain uniformity of component distribution.

The membrane is then contacted with a third solution containing the remainder of the reagent system (the interacting substance). Preferably, absorption of the solution is effected simply by contacting the relatively sparse surface of the membrane with the solution and allowing the membrane to absorb the solution like a blotting paper. This third solution also contains a substance that can be functionally referred to as a "flow control agent." The flow control agent regulates the rate at which the fluid fraction of the sample spreads/distributes throughout the membrane matrix. Thus, flow control agents are effective in preventing separation, such as in chromatography of reagents in IJ hooks, when a fluid sample is added to the membrane. After adding this third solution, the membrane is air-dried to remove excess fluid, lyophilized, and shielded from light. The above-described operations are necessary and appropriate to maintain uniformity of reagent distribution and protect the indicator from destruction by light.

Once the reagent-releasing article is produced as described above, the resulting membrane can be applied to any of several test strip formats specific to the analysis of whole blood, urine, or saliva.

FIG. 1 shows the internal structure of the dry chemical reagent release article of the present invention. The sample receiving surface (2) of the membrane (3) is essentially impermeable to cells and particulate matter. On the contrary, the opposite surface (
4) is relatively porous. Matrix of membrane (3) (
6) is not shown at all in Figure 1, but its inherent porosity is a conditioning agent.

There is a significant decrease from the initial value due to the absorption of indicators, interacting reagents, and flow control agents. The relative density of the original film is therefore effectively increased. The density of the membrane containing the dry reagent system is important for assay performance and membrane uniformity. As previously mentioned, providing controlled drying and shielding the indicator from degradation by light is important for both effective and consistent performance.

Film uniformity is also important in ensuring consistent test strip performance. The cast membrane was found to have more consistent quality and uniformity. Therefore, cast membranes are preferred for the test strips according to the invention.

Figure 2 shows test strips (2) used in the analysis of biological fluids.
This is to show the membrane (3) in 0). Test strips of this configuration are used in protocols for depositing biological samples onto the sample-receiving surface (2) of the membrane, with the aim of absorbing a fluid fraction of the sample into the matrix (6) of the membrane. The detection of indicator molecules by inversion and the relatively high porosity surface of the membrane (
From 4), the color development of the indicator can be observed with the naked eye.

This test strip is also a reflectometer of the type shown in Figure 9.

It can be inserted into a nephelometer (measuring turbidity), a photometer or a fluorescent needle. The reporter molecule is measured and compared to a standard curve for the analyte of interest. The instrument is then used to report values based on observations and comparisons to standards.

In one preferred embodiment of the invention, the dry chemical reagent release article shown in FIGS. Used together to improve detection of analytes of interest. Such improvements can be made separately from the test strip.

Preferably, improvements are made by adding or integrating sample improvement means into the test strip.

In particular, when the dry chemical reagent release articles of the present invention are used in cholesterol assays, a whole blood sample is first absorbed into a porous compressible sponge-like material (i.e., a fiberglass, neoprene, or cellulose pad). was found to be advantageous.

A saline solution containing any of the chemicals or combinations of these materials that enhance the ability of the dry chemical reagent release article to accurately detect the presence of the analyte of interest and/or to quantify this analyte as described above. Process the pad. After the sample is enhanced by contact/interaction/absorption with the pad, the sample is squeezed out of the pad onto the sample receiving surface of the test strip and the reaction of the sample with the dry chemical reagent system proceeds as described above. let

In yet another example of the invention, the dry chemical reagent release article shown in FIGS. 1 and 2 is used in conjunction with a whole blood sample enhancement means to improve the detection of high density lipoprotein (HDL). Since the human body's ability to absorb and/or transport cholesterol depends on the density (molecular weight) of lipoproteins (which are polymeric compounds combined with steroids), total cholesterol and/or low-density lipoproteins (LDL ) is extremely important to measure and quantify HDL. For example, in Example 5, a sample conditioning pad is pretreated with a reagent that precipitates low density lipoproteins. After waiting a short time for this preconditioning interaction to complete, the conditioned sample is expressed onto the sample receiving surface of a cholesterol test strip that has been optimized for increased sensitivity to cholesterol.

The precipitated LDL is trapped along with a portion of the cell fraction of the sample in the preconditioning pad or on the relatively dense surface of the membrane of the test strip, so that the HDL
(primarily) only reaches the dry chemical reagent releasing article within the test strip and initiates a color reaction which is clearly visible on the opposite surface of the test strip. The indicator produced as a result of this reaction can be observed or measured quantitatively and thus can be correlated to the amount of HDL present in the sample.

FIG. 3 is a cross-sectional view of the test strip of FIG. 2 along line A--A. The membrane (3) is enclosed within the test strip components by enclosing members (22, 24), ensuring a confined placement of the sample on the sample receiving surface (2) of the membrane. To encapsulate the membrane within the enclosure in this manner, simply apply a small amount of water-insoluble adhesive (not shown) along the flat surface of the enclosure in contact with the flat surface of the membrane. Second
In the configuration shown in Figures and 4, the sample receiving surface (2) of the membrane (3) is connected to a pair of openings (26
, 28) are exposed above and below. The opening (26) exposing the sample receiving surface (2) of the membrane is indented (70
) determine the horizontal dimension. The sample is this depression (70)
placed within. The limited placement of the sample (99) is better shown in FIG.

A second opening (28) in the enclosure exposes the underside (4) or relatively more porous surface of the membrane, thereby making it possible to monitor reaction products indicative of the presence or absence of the analyte of interest. These reaction products can be observed without physically removing the sample residue from the sample-receiving surface of the membrane or covering the sample residue with a separate “blocking layer” (optical shield). /measurable is unique to the dry reagent release article of the present invention.

The test strips shown in FIGS. 4 and 5 are the same basic components as in the test strip of FIG. 2, with certain improvements. In particular, the test strip in Figure 4 has a protective film (6
0) is added and the protective film (60) is effective to prevent the sample from contacting the underside of the membrane. Therefore,
The test strip is immersed in a fluid sample (i.e.
suitable for contacting the sample with the sample-receiving surface of the membrane (such as a dipstick). Grooves (62) or air bleeds are provided on the surface of the enclosure member to be trapped within a depression (not shown) formed between the protective film (60) and the underside (4) of the membrane. Allow air to escape.

The test strip shown in FIG. 5 has an added closure lid (80), which can be separate or integral with the test strip.

A pressure sensitive adhesive (not shown) is applied to this opening/closing lid.

This allows the user to insert the sample (99) into the recess (70) formed by the opening in the enclosure and the sample receiving surface of the membrane.
) can be effectively encapsulated.

The arrangement in which the sample is thus confined within the test strip provides important protection to the clinician from accidental contact with infectious samples and also protects the indicator from forming within the membrane. Preventing unintentional transfer of sample residue to the surface of an analytical device (i.e., a fluorometer) that can be used for concentration monitoring.

In this latter regard, the test strip is suitable for use in a variety of environments, including use with monitors of the type shown in FIG.

In fact, a test strip (100) having the shape shown in FIG.
104) into a reading station (not shown). This is accomplished by simply lifting the door (106) located on the top of the monitor's housing and positioning the test strip into the monitor's reading station. The test strip is oriented such that the aperture on the underside of the test strip is aligned with the monitor optics (not shown).

During placement and orientation of the test strip, one or more surfaces of the chamber housing and the optical element typically make at least some contact with the test strip. If the sample cannot be isolated as shown in FIG. 5, this will inevitably lead to contamination of this chamber and may damage the monitor or make subsequent sample measurements inaccurate. At required intervals, a monitor measures the indicator level in the test strip. The test strip is then removed from the monitor and discarded.

FIG. 6 shows another example of a surrounding member for use in a test strip according to the invention. The enclosure has multiple openings and is suitable for enclosing separate pieces of membrane containing different dry chemical reagent systems. A device of this configuration can therefore be used to perform clinical chemistry distribution analysis of individual patient samples in one test strip.

FIG. 7 shows an example of a dry chemical reagent release article of the present invention in which a portion of the reagent system is deposited as a thin film or coating (103) on the sample receiving surface of the membrane, and the remainder of the reagent system is present in the matrix of the membrane. show. It is only a temporary condition that the components of the reagent system are separated from each other during the drying stage. When a fluid sample is applied to this coating, the analyte in question is displaced or in any case reacts with a portion of the reagent system, thus allowing this component of the reagent system to be freely absorbed into the membrane; This component of the reagent system is now capable of initiating a discernible reaction that indicates the presence or absence of the analyte of interest.

Figure 8(a) shows a diffusion layer (
120).

The diffusion layer (120) facilitates sample distribution on the sample receiving surface of the membrane and can also be used to move the sample laterally from one portion of the membrane to another.

2. Other Dry Chemical Reagent Release Articles From the above discussion regarding glucose analysis of whole blood, it is easy to manufacture test strips for use with a variety of other analytes found in biological fluid samples and to perform clinical assays. It can be analogized to Accordingly, the conditioned membrane article of the present invention can be applied in the clinical analysis of cholesterol, triglycerides, bilirubin, creatinine, urea and alpha-amylase. The assay format may be essentially the same as described above for glucose, or may include an optionally conditioned membrane/reagent system and one or more additional layers (i.e., diffusion layer, radiation layer, etc.). (blocking layer, semi-transparent diffusion layer, etc.).

Conditioned membranes incorporating dry chemical reagent systems for each of the analytes described above are prepared using essentially the same process described for the production of glucose-specific dry chemical reagent release articles (e.g., the membrane is subjected to a flow control agent). conditioning process and absorbing the indicator/reagent cocktail). Accordingly, conditioning the membrane can be performed prior to or simultaneously with contacting the membrane with one or more components of the dry chemical reagent system.

Test strips for urea can be prepared by imbibing a conditioned membrane with appropriate concentrations of urease, a buffer ring, and an indicator that senses pH changes. When whole blood is brought into contact with the sample receiving surface of the membrane,
Serum is absorbed by the membrane. Urea present in serum is digested by the enzyme urease, releasing ammonia into solution. This ammonia is a suitable indicator (i.e. a protonated merocyanine dye).
can react with. The pH of the membrane is approximately 8.0 with the buffer.
to maintain a relatively low equilibrium concentration of ammonia. The indicator is monitored at 520 nm. A more detailed description of this particular reagent system can be found in publications such as Spade, R.
Spayd RoW et al., "Clinical Chemistry", Volume 24, No. 8 (1978),
It is described on page 1343.

Test strips for α-amylase are prepared by applying a derivatized substrate (i.e.,
It can be produced by absorbing starch) and a buffering agent. When a whole blood sample is deposited on the sample receiving surface of the test strip, serum is absorbed into the membrane and digestion of the derivatized substrate by alpha-amylase in the sample begins. Digestion of such substrates releases chromophores or fluorophores, which can be monitored by conventional techniques and readily available equipment. A more detailed description of this particular reagent is also found in the Spade reference cited above.

Test strips for bilirubin are prepared by imbibing a conditioned membrane with an appropriate concentration of a cationic polymer (i.e., a quaternary salt of the polymer) and a phosphate buffer (pH approximately 7.4). can do. When a whole blood sample is deposited on the sample receiving surface of the test strip,
Serum is absorbed into the membrane and interaction between bilirubin and the cationic polymer begins. As a result of this interaction, the maximum absorption of bilirubin shifts from 440 nm to 45 Q nm, with a concomitant significant increase in absorption at the new peak. A more detailed description of this particular reagent system can also be found in the Sveid reference cited above.

Test strips for triglycerides (triacetylglycerin) are prepared by adding a surfactant to a conditioned membrane.
It can be produced by absorbing lipase, adenosine triphosphate (ATP), glycerol kinase and L-α-glycerol phosphate oxidase, triarylimidazole, and leuco dye. In short, surfactants help dissolve lipoprotein complexes so that lipase can react with triglycerides to produce glycerin and fatty acids. Glycerin is then phosphorylated by adenosine triphosphate in the presence of glycerol kinase enzyme. The mono-α-glycerol phosphate thus produced is then oxidized to dihydroxyacetone phosphate and hydrogen peroxide by L-α-glycerol phosphate oxidase.

The hydrogen peroxide oxidizes the leuco dye to produce a colored indicator with an absorption peak at 640 nm. A more detailed description of reagent systems of this nature is provided in the Spade reference cited above.

Other chemical reagent release articles suitable for the analysis of triglycerides can be made by adsorbing lipase, glycerin dihydrogenase, p-iodonitrotetrozolium violet (INT) and diaphorase onto conditioned membranes. Serum triglycerides interact with the initial chemical reagent system and are hydrolyzed to free glycerin and fatty acids. Free glycerin is then converted to dihydroxyacetone by glycerin dihydrogenase in the presence of NAD.

Simultaneously with such conversion, INT (colorless) is converted to a red dye (up to r = 5) by diaphorase in the presence of NADH.
00 nm). The absorbance of the test strip at 500 nm is directly proportional to the serum triglyceride concentration.

Test strips for measuring total cholesterol in serum can be prepared by adsorbing cholesterol ester hydrolase, cholesterol oxidase, leuco dye and peroxidase onto conditioned membranes.

When a whole blood sample is deposited on the sample-receiving surface of the test strip, serum is absorbed into the membrane and the conversion of cholesterol esters to cholesterol is initiated. Oxides are released.

The peroxide and leuco dye then interact in the presence of peroxidase to produce a highly colored indicator;
This indicator can be monitored visually or by using an instrument. A more detailed description of this particular reagent system can be found in Dappen.

Dappen, G.N., et al., "Clinical Chemistry", Volume 28, No. 5 (198
2), page 1159.

Test strips for creatinine can be prepared by adsorbing appropriate concentrations of creatinine iminohydrolase and an ammonia indicator (ie, bromophenol blue) onto a conditioned membrane. When a whole blood sample is deposited on the sample receiving surface of the test strip,
Serum is absorbed into the membrane and creatinine begins to interact with the enzyme and creatinine aminohydrolase, resulting in the release of ammonia. There, the ammonia reacts with the indicator and the coloring phenomenon is monitored with the naked eye or with conventional equipment. A more detailed description of this particular reagent system can be found in, for example, Tofaretch.

Toffaletti, J. et al., "Clinical Chemistry" Vol. 29, No. 4 (1983)
, page 684. ' The dry chemical reagent release article of the present invention is also available in New York,
It can be used with multilayer test slides of the type developed by Eastman Kodak Company of Rochinister (hereinafter referred to as the "Kodak format").

When a permeable material (i.e., a spreading layer) is placed in adjacent contact with the sample-receiving surface of a treated membrane, such contact affects the velocity and volume of whole blood/fluid moving through the membrane. affect. As a result, the rate and extent of the reaction is mediated by analyte-specific components within the membrane.

At relatively high analyte levels in blood, sample migration across the membrane can result in an excess of analyte, reducing the usable measurement range.

FIG. 7 shows test details according to the present invention in U.S. Pat.
An example is given for use in a displacement immunoassay of the type described in No. 6.232 (Liota). In the article shown in Figure 7, the sample-receiving surface of the membrane is coated with an enzyme-labeled antigen or antibody (hereinafter referred to as an "enzyme-labeled conjugate").

The method of applying the coating to the sample receiving surface is such that the coating material does not penetrate into the membrane matrix. The remainder of the immunochemical reagent system, particularly the luminescent or fluorogenic substrate for the enzyme, is incorporated into the conditioned membrane to keep said substrate separated from the surface coating. Displacement of the enzyme-labeled material occurs upon contacting the sample with the coating on the surface of the membrane.

Displacement of the enzyme-labeled conjugate is based on a dynamic equilibrium due to the presence of the analyte in the sample and competition with the conjugate to bind to the analyte mimic in the surface coating.

The displaced enzyme-labeled conjugate is absorbed into the membrane matrix along with a portion of the fluid fraction of the sample.

The enzyme portion of the conjugate interacts with the enzyme-specific substrate to produce a discernible change in color and fluorescence, which indicates the analyte of interest. This change can be observed with the naked eye or with equipment designed for this purpose.

Section 8(b) shows a novel membrane/wick composite with unique operational advantages. The gauze in this complex has two functions: (a) Opening in gauze (
(b) absorbing excess sample so that the amount of fluid absorbed by the membrane is adjusted and to prevent accidental transfer of the optical surface to other surfaces (particularly optical surfaces within the monitoring equipment shown in FIG. 9). This limited amount of sample absorbed by the membrane results in a limited endpoint reaction that is easy to monitor. This membrane/gauze composite is preferably incorporated into the enclosure (22, 24) of a test strip (20) of the type shown in FIG. The orientation of the composite within the enclosure is the same as the membrane (3) in FIG. The gauze opening (30) of the composite is connected to the opening (30) of the surrounding member (
26). Therefore, when a whole blood sample is deposited on the sample receiving surface of the composite membrane (3),
Blood is drawn essentially uniformly from the blood deposit points on the membrane surface by the gauze (120). By facilitating the distribution of the sample in this manner, the reaction between the analyte and the dry chemical reagent system can proceed more uniformly, thus avoiding uneven color/indicator development.

The presence of a diffusion layer is also important if the diffusion layer is used to move this sample laterally from the point at which the whole blood sample is deposited on the test strip to the reaction site, or if the diffusion layer is used to absorb the sample into the membrane. It may be desirable if the diffusion layer contains reagents for pre-treating or conditioning the sample beforehand. When adjacent layers are used with membranes, the flow through the membrane can be adjusted by varying the concentration of flow modifiers contained in the impregnating vehicle (i.e., polyvinylpyrrolidone, polyethylene glycol, etc.). Can be done. As the concentration of these components increases, the flow rate across the membrane decreases, preserving the usable range of the test strip so that the contiguous layer that acts as a transport agent (contigu
ous wicking layer) is compensated for.

Next, the present invention will be explained with reference to examples. In the examples, parts and percentages mean parts by weight and percentages by weight unless otherwise specified.

Example 1 A dry reagent release article was made according to the method of the present invention using the following materials and reagents: (a) Membrane Millipore (trade name) MF (mixed cellulose acetate-cellulose nitrate and cellulose acetate) Density 4 ,9~6.5■/C-dake 2 Porosity 0. Old ~ 0.45 (sample receiving surface) (Porosity value indicates the difference in pore size from relatively dense to relatively less dense surface) W/V) Aqueous solution Deionized water 〇-Tolidine hydrochloride (c) Reagent cocktail specific for glucose Glucose oxidase (activity 481 (1/-) peroxidase (
Activity 4510/mm 0.1M citrate buffer Enzyme stabilizer - Albumin 0.2% (W/V) Conditioning and flow control agent - Polyvinylpyrrolidone 3% (W/V) The membrane is 305mm wide ( It is commercially available in rolls of 1 foot). To facilitate handling, the membrane can be cut into strips or squares. All of the above solutions were prepared fresh from reagent grade chemicals and deionized water. The membrane was contacted with the indicator solution by first simply dipping the membrane into a trough containing the indicator solution. saturating the membrane with the indicator solution within 30 seconds, then removing the membrane from the indicator solution and removing the solution;
Dry at room temperature or by mild heating.

After the absorption of the indicator was complete, the membrane was contacted exclusively through the relatively large porosity surface with a solution containing a conditioning agent, a flow control agent, and a glucose-specific reagent cocktail. Absorption of this solution by the membrane is accomplished by simply floating the membrane above the solution in a shallow tray, and bringing the relatively large porosity (low density) surface of the membrane onto the surface of the solution, much like applying a flake with a sponge. brought into contact.

Absorption of this solution by the membrane occurs fairly rapidly, usually 3
It was within 0 seconds. The film is then air-dried, residual moisture removed by vacuum drying (lyophilization), and stored for exposure to light and
Protected from the disintegrating effects of oxygen and moisture.

Example 2 The procedure of Example 1 was repeated. However, the order of contacting the membrane with the indicator solution and the membrane with the solution containing the conditioning agent, flow modifier, and glucose-specific reagent cocktail was reversed.

Example 3 The procedure of Example 1 was repeated. However, the membrane was first treated with a solution containing a conditioning agent.

As explained above, the purpose of such treatments is to alter the internal structure of the membrane, converting an essentially passive membrane into one that actively transports solutes. Conditioning the membrane also reduces its porosity and increases the impediment to the throughput of fluid flow.

Thus, the porosity gradient between both surfaces of the membrane is thereby essentially eliminated, and either surface of the membrane acts as a sample receiving surface for a heterogeneous fluid sample.

Solutions containing an indicator solution, a flow modifier, and a glucose-specific reagent cocktail were then sequentially absorbed onto the membrane as described in Example 1.

Example 4 A series of test strips having the shape shown in FIG. 2 were manufactured incorporating the dry chemical reagent release articles of Examples 1.2 and 3, respectively. Each test strip was then evaluated for sample absorption rate, sensitivity, uniformity of indicator color development, and ease of use. During evaluation, simply one or two drops of fresh whole blood were transferred to the sample receiving surface of the membrane. The performance of the test strip was then measured by monitoring the color development of the indicator on the observation side of the membrane.

Test strips incorporating the dry chemical reagent release articles of Examples 1 and 3 both showed well-correlated results over the 50-600 mg/dl range.

Test strips incorporating the dry chemical reagent release article of Example 2 did not show clinically reliable results.

This lack of accuracy in test strips incorporating those obtained in Example 2 is due to the fact that one or more of the enzymes in the reagent cocktail is inactivated by the acidic indicator solution. . The sequential absorption of the components of the reagent cocktail in the membrane is therefore determined by the relative compatibility of the carrier fluid's pH. In the absence of such incompatibility, the order of absorption is not critical, and in fact it is generally possible to absorb all such components from a common solution.

Example 5 A dry chemical reagent release article of the present invention can be used in a cholesterol test strip that is combined with both the configuration shown in FIG. 2 and a sample conditioning pad. This sample conditioning pad is preferably pre-placed over the membrane pores of the test strip shown in FIG. In one commercial example of this article, the sample conditioning membrane is physically integrated into the membrane-containing enclosure.
Make it adhere. A whole blood sample is deposited on this sample conditioning pad. The sample conditioning pad facilitates the release of cholesterol from cholesterol binding proteins in the sample. The sample conditioning pad also increases the degree of physical separation of cellular material from the serum fraction of the sample. However, such physical separation is not considered a prerequisite for effectiveness in releasing cholesterol from binding proteins, nor is it a prerequisite for the ability to squeeze the conditioned sample onto the sample-receiving surface of the test strip. I can't even think of that.

Dry chemical reagent release articles of the invention were prepared from the following materials according to the procedure described in Example 1: (a) Membrane Millipore (trade name) MF (mixed cellulose acetate-cellulose nitrate and cellulose acetate) density 4 .9 to 6.5 ■/C is the porosity of 0.01 to 0.45 (sample receiving surface) (the porosity value is the size of the pores from a surface with relatively high density to a surface with relatively low density (b) Indicator - 1% (W/
V) Aqueous solution Deionized water 0-Tolidine hydrochloride (c) Reagent cocktail specific for cholesterol Cholesterol esterase (activity 40 I mouth/mf) Cholesterol oxidase (activity 4011/rId peroxidase (activity 148 III/-) 0.1M Citrate Buffer Enzyme Stabilizer - Albumin 0.2% (W/V) Conditioning Agent and Flow Control Agent - Polyvinylpyrrolidone 3% (W/V) (d) Aqueous Salt Solution (Saline Solution) Containing Sample Conditioning Agent A sample conditioning pad was manufactured by treating a compressible sample absorbent material using a sample-absorbent material.

The sample conditioning pad was a glass fiber mat (retentive paper classification 0.5-3 μm) and a conditioning agent It)! J) TRITON,
Product Name) X-100 Teared. Other examples of conditioning pads are sponge-like materials made of cellulose or neoprene.1. :100 U of thrombin and physiological saline.

A sample conditioning pad was placed over the holes in the test strip to expose the sample receiving surface of the membrane in the test strip. A whole blood sample was applied to this sample conditioning pad in the same manner as when using other test strips according to the invention.

After the pad has absorbed the sample and the pad-sample interaction is complete (usually within 30-60 seconds), the pad is manually compressed to squeeze the conditioned sample onto the sample-receiving surface of the membrane. Ta. The fluid fraction of the expressed sample was immediately absorbed by the dry chemical reagent release article and interacted with it, resulting in a color reaction. A color reaction shows a correlation between color and the value or level of cholesterol in a sample. I was able to immediately relate it to the color chart/index.

Example 6 In the following immunoassays, the conditioning membrane technology of the present invention was applied to several immunoassays that have traditionally been performed in solution or in a classical heterogeneous format or in a multilayer stacked in-phase format.

1. First, a quantitative amount of peroxidase-labeled β was applied onto the sample-receiving surface of the same type of membrane used in Example 1.
- Test strips were prepared by coating with a thin film containing human chorionic gonadotropin (β-HCG conjugate).The antibody portion of the conjugate was immunochemically applied to the heptene mimic present in this coating. By combining β-HC
The G condidigate was effectively immobilized. Care was taken to ensure that the conjugate was not absorbed into the membrane matrix. After performing such surface coaching, the balance of the reagent system was absorbed into the membrane by absorption from the side with relatively high porosity.

The order of absorption followed the experience gained in previous examples.

That is, the indicator o-)IJ lysine in an acidified vehicle is first absorbed, then the glucose oxidase,
A second solution containing glucose and conditioning agents with citrate buffer was absorbed. The format of the test strip incorporating the dry chemical reagent release article (specific for β-HCG) was the same as that shown in FIG.

Urine samples from freshly defecated pregnant women were collected early in the morning.

The sample was then deposited on the sample receiving surface of the test strip by dipping the test strip into the urine sample. In this way, the β-HCG in the urine sample is brought into contact with the coating on the sample receiving surface of the test strip, and the β-HCG in the urine sample is brought into contact with the coating on the sample receiving surface of the test strip.
A portion of the CG enzyme conjugate was replaced and bound to the analyte. Replacing the conjugate with the analyte allows the conjugate to be absorbed into the test strip where its enzyme moiety reacts with the corresponding substrate (8202) and finally the oxidation of the o-)lysine (and its coloration). (conversion to indicator) occurs. In this way, the presence of the analyte in the sample could be shown by color development.

2. First, add Example 5 to the conditioned membrane.
Test strips suitable for use in competitive enzyme immunoassays were prepared by adsorbing the substrate/color-forming composition.

Additionally, an enzyme (vertoxidase)-labeled antibody (ie, β-HCG) was absorbed into this membrane. A specific quantitative amount of human β-HCG antibody was coated onto the sample receiving surface of the membrane. A urine sample was then deposited on the sample receiving surface of the membrane, and the β-HCG antibody on the surface was allowed to react with the analyte in the sample. Immunochemical interaction between the analyte and the β-HCG antibody prevented absorption of the antibody into the membrane matrix. In this way, the relative concentration of the analyte adjusted the intensity of the indicator's color development, with higher concentrations weakening the intensity of the color development. The degree of color development could be monitored visually or by using equipment.

Other indicator systems included fluorescently labeled conjugates (FIA) or radioisotope labeled conjugates (RIA). In each of these other assay formats, indicator concentration was monitored by the instrument.

3. Leveling the conditioned membrane assay format of the present invention by simply absorbing β-HCG labeled colloidal gold particles into the membrane.
ing) (US Pat. No. 4,313,734) was applied. A quantitative amount of this reagent was applied to the membrane and dried to impart a uniform color to the membrane surface. An aliquot of the sample (ie, urine) was then deposited on the sample receiving surface of the membrane. The presence of antibody in the sample was evidenced by aggregation of the β-HCG labeled colloidal gold particles, ie, a discernible color change that could be observed with the naked eye.

[Brief explanation of the drawing]

1 is a perspective view of a portion of an example of a dry chemical reagent release article of the present invention; FIG. 2 is a perspective view of a portion of the interior of a test strip for whole blood sample analysis incorporating the article of FIG. 1; FIG. 3 is a sectional view taken along the line A-A in FIG. 2; FIG. 4 is a partially internal view of a test strip for urine sample analysis incorporating the article shown in FIG. 1; 5 is a perspective view showing a part of the inside of another example of the test strip shown in FIG. 2, and FIG. FIG. 7 is a perspective view showing a part of another example of the dry chemical reagent release article of the present invention; FIG. 8(a) and FIG. FIG. 9 is a perspective view of a portion of yet another example of a reagent-releasing article; FIG. FIG. 2... Sample receiving surface 3... Membrane 4... Opposite surface, lower side (surface with relatively high porosity) 6... Membrane matrix 20... Test strips 22, 24... - Surrounding member 26.28... Opening 30... Opening 60... Protective film 62... Groove 70... Hollow 80.
...Opening/closing lid 99...Sample 100...Test strip 103...Thin film (coating) 104...
・Monitor 106...Door 120...Diffusion layer (gauze)

Claims (1)

Claims: 1. In making a dry chemical reagent release article suitable for the analysis of heterogeneous fluid samples, the method comprises: (a) providing an absorbent film having an essentially uniform composition; having two flat surfaces and a porosity gradient from one flat surface to the other flat surface, at least one of said flat surfaces containing cells present in a biological fluid sample; (b) said film has an inherent porosity that is essentially impermeable to particulates on the order of the size of; (c) contacting said absorbent film with an absorption effective amount of a conditioning agent, said conditioning agent improving the absorption properties of said film for said fluid sample, said film and said conditioning agent; A method for producing a dry chemical reagent releasing article, characterized in that the contacting with the film is carried out independently before the contact between the film and the indicator, or after the contact between the film and the indicator. 2. The absorbent film is coated with an effective amount of albumin for absorption;
2. The method of claim 1, further comprising conditioning with one conditioning agent selected from the group consisting of polyvinylpyrrolidone, polyethylene glycol, carbohydrates, (water-soluble) carboxymethylcellulose, methylcellulose, glycerin, and polyoxyethylene ether. 3. The method of claim 1, wherein the absorbent cast film is selected from the group consisting essentially of cellulose acetate-cellulose nitrate, nylon, and polysulfone. 4. The method of claim 1, wherein the reagent cocktail is specific for glucose. 5. The method of claim 1, wherein the reagent cocktail is specific for cholesterol. 6. The method of claim 1, wherein the reagent cocktail is specific for urea. 7. The method of claim 1, wherein the reagent cocktail is specific for antigen or antibody detection. 8. The method of claim 7, wherein the reagent cocktail contains an antigen or antibody that binds to the analyte mimic. 9. The method of claim 7, wherein the reagent cocktail is specific for the detection of hormones, drug compounds, drugs of abuse or protein-containing substances capable of eliciting an immune response. 10. depositing a portion of the reagent cocktail onto one of the planar surfaces of the absorbent film and retaining it on the one of the planar surfaces without substantial penetration into the matrix of the absorbent film; 8. The method according to claim 7. 11. A dry chemical reagent release article suitable for detecting analytes in a heterogeneous fluid sample, said article having an essentially uniform composition and a porosity extending from one planar surface to another. comprising a gradient absorbent film, the inherent fluid absorption-distribution properties of which have been modified by sequential absorption of a conditioning agent, an indicator, a flow control agent, and a reagent cocktail into the matrix; provides an essentially uniform distribution of indicators, flow modifiers, and reagent cocktails within the absorbent film, thereby improving the uniformity of the internal structure of the film to improve the uniformity of absorption, and A dry chemical reagent release article characterized in that the dry chemical reagent release article is configured to adjust the absorption rate of and the interaction of the fluid sample with the reagent cocktail. 12. The absorbent film is conditioned with an absorption effective amount of one conditioning agent selected from the group consisting of albumin, polyvinylpyrrolidone, polyethylene glycol, carbohydrates, carboxymethylcellulose, methylcellulose, glycerin, and polyoxyethylene ether. Article according to item 11. 13. The article of claim 11, wherein the absorbent cast film is selected from the group consisting essentially of cellulose acetate-cellulose nitrate ester, nylon, and polysulfone. 14. The article of claim 11, wherein the reagent cocktail is specific for glucose. 15. The article of claim 11, wherein the reagent cocktail is specific for cholesterol. 16. The article of claim 11, wherein the reagent cocktail is specific for urea. 17. The article of claim 11, wherein the reagent cocktail is specific for antigen or antibody detection. 18. The article of claim 17, wherein the reagent cocktail contains an antigen or antibody that binds to the analyte mimic. 19. The article of claim 17, wherein the reagent cocktail is specific for the detection of hormones, drug compounds, drugs of abuse, or protein-containing substances capable of eliciting an immune response. 20. depositing a portion of the reagent cocktail onto one of the planar surfaces of the absorbent film and retaining it on the one of the planar surfaces without substantial penetration into the matrix of the absorbent film; The article according to claim 17. 21. In screening heterogeneous fluid samples for rapid analysis of analytes of interest, (a) essentially homogeneous composition and porosity gradient from one flat surface to the other; The unique fluid absorption-distribution properties of the film are modified by sequential absorption of a conditioning agent, an indicator, a flow control agent, and a reagent cocktail into its matrix. The effect of such absorption is to provide an essentially uniform distribution of indicators, flow modifiers, and reagent cocktails within the absorbent film, thereby improving the uniformity of the internal structure of the film and improving the uniformity of the fluid sample. (b) adjusting both the rate of absorption and the uniformity of absorption and the interaction of the fluid sample with the reagent cocktail; (b) applying the sample receiving surface of the absorbent film of the reagent release article to the (c) monitoring a change in color or fluorescence or development of color or fluorescence on a surface of said absorbent film opposite said sample-receiving surface; Method for analyzing homogeneous fluid samples. 22. The absorbent film is conditioned with an absorption effective amount of one conditioning agent selected from the group consisting of albumin, polyvinylpyrrolidone, polyethylene glycol, carbohydrates, carboxymethylcellulose, methylcellulose, glycerin, and polyoxyethylene ether. The method according to item 21. 23. The method of claim 21, wherein the absorbent cast film is selected from the group consisting essentially of cellulose acetate-cellulose nitrate, nylon, and polysulfone. 24. The method of claim 21, wherein the reagent cocktail is specific for glucose. 25. The method of claim 21, wherein the reagent cocktail is specific for cholesterol. 26. The method of claim 21, wherein the reagent cocktail is specific for urea. 27. The method of claim 21, wherein the reagent cocktail is specific for antigen or antibody detection. 28. The method of claim 27, wherein the reagent cocktail contains an antigen or antibody that binds to the analyte mimic. 29. The method of claim 27, wherein the reagent cocktail is specific for the detection of hormones, drug compounds, drugs of abuse or protein-containing substances capable of eliciting an immune response. 30. depositing a portion of the reagent cocktail onto one of the planar surfaces of the absorbent film and retaining it on the one of the planar surfaces without substantial penetration into the matrix of the absorbent film; 28. The method of claim 27. 31, in a test kit for the analysis of heterogeneous fluid samples, comprising: (a) an absorbent film having an essentially uniform composition and a porosity gradient from one flat surface to another flat surface; Unique fluid absorption and distribution properties include conditioning agents, indicators,
modified by sequential absorption of a flow control agent and a reagent cocktail, the action of such absorption resulting in an essentially uniform distribution of the indicator, flow control agent and reagent cocktail within said absorbent film, thereby increasing the concentration of said film. (b) further, improving internal structure uniformity to tune both the rate of absorption and the uniformity of absorption of the fluid sample and the interaction of the fluid sample with the reagent cocktail; A test kit comprising a standard for comparison with the indicator of the dry chemical reagent releasing article. 32. The absorbent film is conditioned with an absorption effective amount of one conditioning agent selected from the group consisting of albumin, polyvinylpyrrolidone, polyethylene glycol, carbohydrates, carboxymethylcellulose, methylcellulose, glycerin, and polyoxyethylene ether. The test kit according to item 31. 33. The Tetos kit of claim 31, wherein the absorbent cast film is selected from the group consisting essentially of cellulose acetate-cellulose nitrate, nylon, and polysulfone. 34. The test kit of claim 31, wherein the reagent cocktail is specific for glucose. 35. The test kit of claim 31, wherein the reagent cocktail is specific for cholesterol. 36. The test kit of claim 31, wherein the reagent cocktail is specific for urea. 37. The test kit of claim 31, wherein the reagent cocktail is specific for the detection of antigens or antibodies. 38. The test kit of claim 37, wherein the reagent cocktail contains an antigen or antibody that binds to the analyte mimic. 39. The test kit of claim 37, wherein the reagent cocktail is specific for the detection of hormones, drug compounds, drugs of abuse or protein-containing substances capable of eliciting an immune response. 40, depositing a portion of the reagent cocktail onto one of the planar surfaces of the absorbent film and retaining it on the one of the planar surfaces without substantial penetration into the matrix of the absorbent film; 38. The Tetos kit according to claim 37.