Apparatus and method for detecting pancreatic lipase

Abstract

A method and device for determining pancreatic lipase in an animal sample is provided. In one aspect, the device includes a basic buffer layer containing one or more bile salts and calcium; a separator layer containing colipase; and an indicator layer containing a chromogenic substrate such as DGGR. The device may also include a system for optically determining the amount of color change that occurs. The method includes using the device to detect pancreatic lipase in an animal sample. In another aspect, the present disclosure provides a kit including the device described in various aspects and embodiments herein and instructions for use of the device.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 435,766, filed December 28, 2022, which is incorporated herein by reference in its entirety.

[0002] Field The present disclosure relates to devices and methods for determining the presence and / or quantity of pancreatic lipase in a sample, which may be used in the diagnosis and management of pancreatitis in animals. [Background technology]

[0003] background Lipase is a glycoprotein triglyceride hydrolase that catalyzes the cleavage of triglycerides into diglycerides, with the subsequent formation of monoglycerides and fatty acids.Excessive serum lipase is a symptom of pancreatitis, a pancreatic disease characterized by excessive lipase, protease and amylase production.Excessive lipase production can then lead to a variety of symptoms, ranging from mild discomfort to death, depending in part on the severity of the disease and the degree of lipase overproduction.

[0004] Pancreatic lipase has been an important clinical chemistry parameter for the differential diagnosis of pancreatic diseases for many years.Many methods for detecting lipase, including enzyme assays, have been described.However, these assays generally have a low correlation between the lipase activity determined in serum and the degree of pancreatic damage.In particular, it has been found that enzyme assays for lipase are not specific enough to distinguish lipase activity from organs other than the pancreas.

[0005] A colorimetric test specific for human pancreatic lipase has been described, based on the cleavage of the chromogenic lipase substrate 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methyl-rsorufin) ester (DGGR). For example, a lipase colorimetric assay reagent is available from Roche Diagnostics, GmbH (Mannheim, Germany). This assay is sold as a kit with two separate reagents. The first reagent contains a buffer and DGGR substrate, along with a bile acid (taurodeoxycholate). The second reagent contains a buffer, colipase, and cholate. Pancreatic lipase activity is specifically determined by the combination of bile acid and colipase used in the assay. Lipase activity is virtually undetectable in the absence of colipase. Colipase activates only pancreatic lipase, but not other lipolytic enzymes found in serum. The high amount of cholate ensures that esterases present in serum do not react with the chromogenic substrate due to the highly negative surface charge.

[0006] The DGGR substrate is cleaved by the catalytic action of alkaline lipase solution to form 1,2-O-dilauryl-rac-glycerol and an unstable intermediate, glutaric acid-(6-methylresorufin) ester, which spontaneously decomposes in alkaline solution to form glutaric acid and methylresorufin. The color intensity of the red pigment formed is directly proportional to lipase activity and can be determined photometrically.

[0007] Other commercially available lipase assays include the Lipase Color Liquid Assay from Sentinel Diagnostics (Milan, Italy) and the Coloripase Coloriometric Assay from Nuclin Diagnostics, Inc. (Northbrook, Illinois).

[0008] The optimal time for measuring serum lipase is approximately 14 days after the onset of acute pancreatitis, with peak lipase activity reached within 24 hours and declining after 8 to 14 days. In animals, symptoms of pancreatitis are generally nonspecific, with vomiting being the most common symptom presented to clinicians. However, this symptom is indicative of many other diseases. Currently, animal testing for pancreatic lipase is typically performed in reference laboratories away from clinics, which delays diagnosis and therefore treatment. A rapid, specific, clinically-based method for pancreatic-specific lipase would enable rapid diagnosis and treatment of patients. Summary of the Invention [Means for solving the problem]

[0009] overview One aspect of the present disclosure provides a device for detecting the presence or amount of pancreatic lipase in an animal sample, the device comprising: a basic buffer layer; a separation layer comprising colipase; an indicator layer comprising a chromogenic substrate, e.g., 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methyl-resorufin) ester (DGGR); and a system for optically determining the amount of color change development in a mixture of the sample, the colipase, and the chromogenic substrate.

[0010] In another aspect, the present disclosure provides a method for detecting the presence or amount of pancreatic lipase in an animal sample, the method comprising: providing an apparatus comprising: (a) a basic buffer layer; (b) an isolation layer comprising colipase; (c) an indicator layer comprising a chromogenic substrate, e.g., DGGR; and (d) a system for optically determining the amount of color change occurring in a mixture of at least the sample, the colipase, and the chromogenic substrate; introducing the sample onto the basic buffer layer for diffusion through the basic buffer layer, the isolation layer, and the indicator layer; determining the color change in the apparatus; and correlating the color change with the presence or amount of pancreatic lipase in the sample.

[0011] In another aspect, the present disclosure provides kits that include a device described in various aspects and embodiments herein and instructions for use of the device.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS Further features, objects and advantages will become more readily apparent from a consideration of the following detailed description, which refers to the following figures. [Brief explanation of the drawings]

[0013] [Figure 1] 1A, 1B, 1C, 1D, 1E, and 1F show an exemplary arrangement of layers for detecting pancreatic lipase of the present disclosure.

[0014] [Figure 2] FIG. 2A shows the percent particle size distribution for emulsified DGGR particles, and FIG. 2B shows the correlation function graph, which indicates low polydispersity with steep traces and nearly perfect overlap for the three runs.

[0015] [Figure 3A] Figure 3A shows that the response from an analyzer using a fully coated dry slide format shows good separation and correlation with the Spec cPL (canine) immunoassay from the applicant, and Figure 3B shows good separation and correlation with the Spec fPL (feline) immunoassay from the applicant. [Figure 3B] Figure 3A shows that the response from an analyzer using a fully coated dry slide format shows good separation and correlation with the Spec cPL (canine) immunoassay from the applicant, and Figure 3B shows good separation and correlation with the Spec fPL (feline) immunoassay from the applicant.

[0016] [Figure 4]FIG. 4 shows the results of storage stability testing of dried slides herein frozen at −20° C. for 0 days, 15 days, 1 month, 3 months, and 6 months; no statistically significant changes in calculated concentrations were observed over the 6-month storage period. DETAILED DESCRIPTION OF THE INVENTION

[0017] While the methods and compositions of the present invention are susceptible to various modifications and alternative forms, exemplary embodiments thereof have been shown by way of example in the drawings and are described in detail herein. It should be understood, however, that the description of the exemplary embodiments is not intended to limit the devices and methods to the particular forms disclosed; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the devices and methods as defined by the embodiments herein and the appended claims. Accordingly, reference should be made to the various aspects and embodiments of this specification and the appended claims to interpret the scope of the devices and methods.

[0018] explanation In one aspect, the present disclosure provides devices and methods for detecting the presence or amount of pancreatic lipase in an animal sample. An embodiment of the device includes a multi-layer slide that allows sample flow through from the top layer to the bottom layer. During sample flow through the layers, a color change in the sample / reagent mixture resulting from the interaction of the pancreatic lipase in the sample with the reagent can be detected visually or with an appropriate instrument.

[0019] In some embodiments, the multi-layer device comprises, for example, (a) a basic buffer layer; (b) a separator layer containing colipase; and (c) an indicator layer containing a chromogenic substrate. The presence, amount, or rate of color change can be detected in the indicator layer. The color can be detected visually, or the device can further comprise or be used in a system for optically determining the color or amount of change.

[0020] Samples suitable for use in the device include biological samples such as tissues or fluids from humans or animals, including, but not limited to, whole blood, plasma, and serum. Many such samples require processing, e.g., dilution, before analysis. Samples include both unprocessed and / or processed samples. In some embodiments, the animal sample is a canine or feline sample.

[0021] With respect to the basic buffer layer, in some embodiments, the basic buffer layer comprises a basic buffer, which in some embodiments has a pH between about (as used in this disclosure, "about" refers to + / - 5%) pH 7 to 10, pH 7 to 9, pH 7 to 8, pH 8 to 10, pH 8 to 9, pH 7.5 to 10, pH 7.5 to 9.5, pH 7.5 to 9, pH 7.5 to 8.5, pH 8 to 10, pH 8 to 9.5, pH 8 to 9, or any one of about pH 7, pH 7.5, pH 8, pH 8.4, pH 9, pH 9.5, or pH 10. In some embodiments, the basic buffer layer is dried in a buffer between about pH 7 to 9.

[0022] Suitable basic buffers include, for example, MOPS (3-(N-morpholino)propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), MOBS (4-(N-morpholino)butanesulfonic acid), DIPSO (3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid), TAPSO (N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid), TEA (triethanolamine), pyrophosphate (pKa 4), HEPPSO (N-(2-hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid), POPSO (piperazine-N,N'-bis(2-hydroxypropane) sulfonic acid), Tricine, Hydrazine, Glycylglycine (pKa 2), Trizma (Tris), EPPS (N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid)), HEPPS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), BICINE or Bicine (N,N-bis(2-hydroxyethyl)glycine), HEPBS (4-[4-(2-hydroxyethyl)piperazin-1-yl]butane-1-sulfonic acid), TAPS (3-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}propane-1-sulfonic acid), AMPD (2-amino-2-methyl-1,3-propanediol), TABS (N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid), AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid), taurine (AES), borate (pKa 1, pKa 2 or pKa 3), CHES (2-(cyclohexylamino)ethane-1-sulfonic acid), AMP (2-amino-2-methyl-1-propanol), glycine (pKa 2), ammonium hydroxide, CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), carbonate (pKa 2), methylamine, piperazine (pKa 2), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), and others suitable for supporting biological reactions at basic pH levels. pKa1 represents the negative base 10 logarithm of the first (lowest) acid dissociation constant (Ka) of a solution, and pKa2 (pKa3, etc.) represents the negative base 10 logarithm of the second (third, etc.) acid dissociation constant (Ka) of a solution when two (three, etc.) dissociations are possible. Appropriate pH buffering behavior is generally observed within + / - 1 unit of the pKa value; therefore, buffers with pKa values ​​of 7 or greater may be suitable for the basic buffer layer.

[0023] In certain embodiments, the basic buffer layer comprises a bicine buffer at pH 8.4. Generally, the amount of basic buffer material is 1-50 mg / ml, for example, 5-45 mg / ml, 10-30 mg / ml, or 15-25 mg / ml.

[0024] In some embodiments, the basic buffer layer may further comprise calcium, which is typically provided as calcium ions in the form of calcium salts including, for example, calcium chloride, calcium carbonate, calcium citrate, calcium lactate, calcium sulfate, calcium malate, and the like.

[0025] In some embodiments, the basic buffer layer further comprises colipase, which may be present at a concentration of about 1 mg / L to about 50 mg / L, e.g., 5 to 45 mg / L, 10 to 30 mg / L, or 15 to 25 mg / L. In some embodiments comprising colipase in the basic buffer layer, the isolation layer does not contain colipase.

[0026] In some embodiments, the basic buffer layer further comprises one or more surfactants. Suitable surfactants can include those having a carboxyl group, a sulfonic acid group, a sulfate group, or a phosphate group as a hydrophilic group. Anionic surfactants having a sulfonic acid group include alkyl benzene sulfonates (e.g., sodium dodecylbenzene sulfonate; SDBS), alkyl naphthalene sulfonates, alkyl sulfates, polyoxyethylene alkyl ether sulfates, α-olefin sulfonates, and N-acylmethyl taurine salts. The anionic surfactant preferably should not inhibit lipase activity or inactivate any enzymes added to the device described herein or utilized in any of the methods described herein.

[0027] In some embodiments, the surfactant is an alkylbenzenesulfonate having an alkyl chain containing 10 to 14 carbon atoms, and in some embodiments, sodium dodecylbenzenesulfonate is used. The salt is most commonly a sodium salt; however, potassium or lithium salts can also be used. The surfactant can be used at a concentration of about 1 g / L to about 6 g / L.

[0028] In some embodiments, the one or more surfactants comprise nonionic surfactants such as ethoxylates, fatty alcohol ethoxylates, alkylphenol ethoxylates (APEs or APEOs), fatty acid ethoxylates, ethoxylated amines and / or fatty acid amides, end-blocked ethoxylates, fatty acid esters of polyhydroxy compounds, fatty acid esters of glycerol, fatty acid esters of sorbitol, fatty acid esters of sucrose, alkyl polyglucosides, etc. In some embodiments, nonionic surfactants according to the present disclosure have a hydrophilic-lipophilic balance (HLB) ranging from about 15 to 18, and the HLB can be calculated by the formula HLB=20*M h / M(where M hHLB is a measure of the degree of hydrophilicity or lipophilicity of a surfactant molecule, determined by calculating the percentage of molecular weight for the hydrophilic and lipophilic portions of the molecule according to (where M is the molecular mass of the hydrophilic portion of the molecule and M is the molecular mass of the entire molecule), and gives a result on a scale of 0 to 20. An HLB value of 0 corresponds to a completely lipophilic / hydrophobic molecule, and a value of 20 corresponds to a completely hydrophilic / lipophobic molecule. In some embodiments, the nonionic surfactant is Tergitol. TM , e.g., Tergitol TM 15S-30 (Dow Chemicals).

[0029] In some embodiments, the nonionic surfactant is soluble in a polar solvent, which in some embodiments is an alcohol, such as methanol, ethanol, n-propanol, isopropanol, n-butanol, and the like.

[0030] In some embodiments, the basic buffer layer further comprises one or more components that facilitate sample propagation and absorption. In some embodiments, the one or more components include polymeric binders (e.g., polyacrylamide), viscosity modifiers (e.g., carboxymethylcellulose), plasticizers (e.g., polyethylene glycol), biocidal components (e.g., Proclin), lubricants, antifoaming agents (e.g., emulsified silicones), particles (or "pigments") ranging in size from 1 to 100 μm, and combinations thereof, as known in the art, e.g., U.S. Pat. No. 3,992,158, U.S. Pat. No. 4,258,001, and U.S. Pat. No. 4,670,381.

[0031] The relative concentration of binder (and optionally viscosity modifiers, plasticizers, biocides, lubricants, antifoaming agents, etc.) to elements that do not change morphology (such as melt flow / film morphology) or evaporate during the coating and drying process is defined by pigment volume concentration (PVC). PVC is a well-known measurement in coating formulations to determine how much binder and "pigment" (where "pigment" is defined as a particle, filler, or other element that does not change during the coating and drying process) should be incorporated into the formulation to achieve a specific porosity in the coating. Typically, porous coatings have a pigment volume concentration greater than 85%, defined by the formula PVC = [ΣVp / (ΣVp+ΣVb)] × 100%, where ΣVp is the sum of the volumes of all "pigments" in the system and ΣVb is the sum of the volumes of all binders in the system. Those skilled in the art know that a compatible binder / particle system should be selected, where the binder's properties are compatible with the surface chemistry of the "pigments" (particles, fillers, etc.).

[0032] As used herein, a PVC in the range of about 87% to 97% (inclusive) is suitable for the applications described herein for binders used in coatings having average or greater strength, with weaker binders capable of supporting even lower PVC than stronger binders. In certain embodiments, a preferred PVC is about 93%. Binder strength also influences the robustness of the coating to manufacturing stresses.

[0033] In some embodiments, the basic buffer layer is in the form of a membrane. For example, a semipermeable membrane (e.g., an IPOC membrane (International Point of Car, Inc., Toronto, Canada)) is coated or impregnated with the components of the basic buffer layer (basic buffer and, optionally, surfactant, colipase, calcium, or bile salts, either separately or in combination), dried, laminated to a separator layer, etc. Preferred semipermeable membranes have consistently sized pores and / or a regular porous structure that act to facilitate sample propagation and absorption in addition to providing buffering capacity from the basic buffer components.

[0034] For the separating layer, the colipase may be present at a concentration of about 1 mg / L to about 50 mg / L, such as 5-45 mg / L, 10-30 mg / L, or 15-25 mg / L.

[0035] In some embodiments, the isolation layer further comprises a support matrix. The support matrix comprises at least one hydrophilic polymer, and in some embodiments, the support matrix comprises a mixture of two or more hydrophilic polymers. The hydrophilic polymer is water-soluble or water-swellable and can be natural, synthetic, or semi-synthetic. Hydrophilic polymers of the present disclosure include starch, pullulan, pullulan derivatives, cellulose, cellulose derivatives (e.g., carboxymethylated cellulose, methylated cellulose, hydroxyethylated cellulose, and hydroxypropylated cellulose), scleroglucan, elsinan, levan, alternants, dextran, agarose, gelatin (e.g., acid-treated gelatin and deionized gelatin), gelatin derivatives (e.g., phthalated gelatin and hydroxyacrylate-grafted gelatin), acrylamide polymers, such as copolymers containing acrylamide and various vinyl monomers, vinylpyrrolidone polymers (e.g., polyvinylpyrrolidone), and acrylate polymers.

[0036] The hydrophilic polymers of the present disclosure may be uncrosslinked or crosslinked with crosslinking agents such as polycarboxylic acids, halogenated dicarboxylic acids, polycarboxylic anhydrides, aldehyde compounds, N-methylol compounds, isocyanate compounds, metaphosphates, divinyl compounds, bisaziridines, etc. The crosslinking may also be unipolymeric (one polymer, whether alone or after crosslinking one portion of a mixture of two or more hydrophilic polymers), dipolymeric (both polymers of a mixture of two polymers are crosslinked in the presence of each other), or multipolymeric (all polymers in a mixture of three or more polymers are crosslinked in the presence of each other, or a subset of two or more polymers in a mixture of two or more polymers plus one polymer are crosslinked before mixing with the remaining polymers).

[0037] A mixture of two or more hydrophilic polymers can have equal proportions of each polymer (e.g., as measured by mass, moles, volume, or other measure), or an excess of one or more polymers relative to the other. The ratio of one polymer to another in a mixture of two polymers can be, for example, about 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, and ratios therebetween. In a mixture of three or more polymers, each can vary in proportion relative to the other polymers in a manner similar to the mixture of two polymers described above. That is, one or more polymers can be in excess of any other polymer, or all polymers can have equal proportions (e.g., by mass, moles, volume, or other measure). For example, a mixture of three polymers (A:B:C) can have ratios of 5:2:1, 1:1:1, 3:10:1, 4:9:1, 1:15:3, 20:1:7, etc.

[0038] In some embodiments, the support matrix is ​​a mixture of cellulose and pullulan, which can have equal proportions of pullulan and cellulose, an excess of pullulan to cellulose, or an excess of cellulose to pullulan. The cellulose to pullulan ratio can be, for example, about 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, and ratios therebetween. In some embodiments, the cellulose to pullulan ratio is 2:1. The support matrix reagent can comprise up to 30% by weight of the final formulation.

[0039] In some embodiments, the isolation layer further comprises an acidic buffer. In some embodiments, the pH is between about pH 1 to 6, pH 1 to 5, pH 2 to 7, pH 2 to 6, pH 3 to 6, pH 4 to 6, pH 2 to 5, pH 3 to 5, pH 2.5 to 5.5, pH 2.5 to 5, pH 2.5 to 4.5, pH 3 to 5.5, pH 3.5 to 5.5, pH 3.5 to 5, or about any one of pH 2.5, pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, or pH 6. In certain embodiments, the pH is between about pH 3.5 to pH 4.5, and in other certain embodiments, the pH of the indicator layer is about pH 4.

[0040] DGGR is unstable under neutral or basic pH conditions. Nevertheless, prior art dry slides traditionally place a layer with a neutral or basic pH adjacent to the DGGR-containing layer, which can lead to, for example, diffusion at the layer interface and an increase in the pH of the DGGR-containing layer. An increase in pH over time can lead to instability of DGGR and the generation of false-positive resorufin-based color. In various embodiments of the present disclosure, DGGR can be maintained at an acidic pH of about 3.5 to about 4.5 until its reaction with pancreatic lipase from an animal sample by providing an isolation layer containing an acidic buffer between the basic buffer layer and the indicator layer (either with or without a separate diffusion layer). This isolation layer with its acidic pH buffer further isolates the DGGR chromogenic substrate from the basic pH conditions of the basic buffer layer during manufacturing and storage, thereby preventing or minimizing false-positive pancreatic lipase activity. Only when an animal sample is actively added to the basic buffer layer does the basic pH buffer from the basic buffer layer diffuse downward through each layer to the indicator layer, raising the pH and supporting pancreatic lipases that require a basic pH for optimal activity. In embodiments where the indicator layer is above the basic buffer layer and an isolation layer is present between them, only when an animal sample is actively added to the indicator layer does the acidic pH buffer from the indicator layer diffuse downward through each layer to the basic buffer layer, raising the pH and supporting any pancreatic lipases in the sample. DGGR can be used at concentrations of about 0.2 g / L to about 10.0 g / L, e.g., about 0.3 g / L to about 9.0 g / L, or about 0.35 g / L to about 8 g / L, or about 0.4 g / L to about 7.5 g / L, or about 0.5 g / L to about 7 g / L.

[0041] Suitable acidic buffers include, for example, maleate (pKa1), phosphate (pKa1), glycine (pKa1), citrate (pKa1), glycylglycine (pKa1), malate (pKa1), formate, citrate (pKa2), succinate (pKa1), acetate, propionate, malate (pKa2), pyridine, piperazine (pKa1), cacodylate, succinate (pKa2), MES (2-(N-morpholino)ethanesulfonic acid), citrate (pKa3), maleate (pKa2), Bis-Tris, carbonate (pKa1). ), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), tartrate, and others suitable for supporting biological reactions at acidic pH levels. Appropriate pH buffering behavior is generally observed within + / - 1 unit of the pKa value; therefore, buffers with pKa values ​​less than 7 may be suitable for the isolation layer. Generally, the amount of acidic buffer substance is 1-50 mg / ml, e.g., 5-45 mg / ml, 10-30 mg / ml, or 15-25 mg / ml.

[0042] In some embodiments, one or more layers of the devices described herein, such as the isolation layer (and in some embodiments, the indicator layer and basic buffer layer), comprise one or more bile salts (i.e., salts of bile acids). Bile salts include, for example, cholate, glycocholate, taurocholate, deoxycholate, taurodeoxycholate, chenodeoxycholate, glycochenodeoxycholate, taurochenodeoxycholate, and lithocholate. Sodium and potassium salts are most commonly observed, with sodium salt being the most common counterion. In some embodiments, the bile salt comprises sodium cholate, sodium deoxycholate, sodium taurocholate, sodium taurodeoxycholate, sodium deoxytaurocholate, or a mixture of two or more thereof; in some embodiments, the bile salt is sodium taurodeoxycholate. In some embodiments, the isolation layer further comprises one or more bile salts. Bile salts can be used at a concentration of about 2 g / L to about 30 g / L, for example, 5 to 25 g / L or 10 to 20 g / L.

[0043] In some embodiments, the isolation layer further comprises one or more surfactants. Suitable surfactants may include those having a carboxyl group, a sulfonic acid group, a sulfate group, or a phosphate group as a hydrophilic group. Anionic surfactants having a sulfonic acid group include alkyl benzene sulfonates (e.g., sodium dodecylbenzene sulfonate; SDBS), alkyl naphthalene sulfonates, alkyl sulfates, polyoxyethylene alkyl ether sulfates, α-olefin sulfonates, and N-acylmethyl taurine salts. The anionic surfactant preferably does not inhibit lipase activity or inactivate any enzymes added to the device described herein.

[0044] In some embodiments, the surfactant is an alkylbenzenesulfonate having an alkyl chain containing 10 to 14 carbon atoms, and in some embodiments, sodium dodecylbenzenesulfonate is used. The sodium salt is the most common salt, but potassium or lithium salts can also be used.

[0045] In some embodiments, the one or more surfactants comprise the nonionic surfactants described above. In some embodiments, the nonionic surfactants according to the present disclosure have a hydrophilic-lipophilic balance (HLB) ranging from about 15 to 18, and in some embodiments, the nonionic surfactants are soluble in polar solvents, such as, but not limited to, alcohols, such as methanol, ethanol, n-propanol, isopropanol, and n-butanol.

[0046] In some embodiments, the isolation layer further comprises a polyvinylpyrrolidone support matrix and a non-ionic surfactant having an HLB between 15 and 18 that is also soluble in alcohol solvents.

[0047] With respect to the indicator layer, in some embodiments, the indicator layer further comprises a support matrix. Similar to the isolation layer, the support matrix for the indicator layer comprises at least one hydrophilic polymer. In some embodiments, the support matrix comprises a single hydrophilic polymer, and in some embodiments, the support matrix comprises a mixture of two or more hydrophilic polymers. The hydrophilic polymer dissolves in or is swollen by water and can be natural, synthetic, or semi-synthetic. Hydrophilic polymers of the present disclosure include starch, pullulan, pullulan derivatives, cellulose, cellulose derivatives (e.g., carboxymethylated cellulose, methylated cellulose, hydroxyethylated cellulose, and hydroxypropylated cellulose), scleroglucan, elsinan, levan, alternants, dextran, agarose, gelatin (e.g., acid-treated gelatin and deionized gelatin), gelatin derivatives (e.g., phthalated gelatin and hydroxyacrylate-grafted gelatin), acrylamide polymers, such as copolymers comprising acrylamide and various vinyl monomers, vinylpyrrolidone polymers (e.g., polyvinylpyrrolidone), and acrylate polymers.

[0048] The hydrophilic polymers of the present disclosure may be uncrosslinked or crosslinked with crosslinking agents such as polycarboxylic acids, halogenated dicarboxylic acids, polycarboxylic anhydrides, aldehyde compounds, N-methylol compounds, isocyanate compounds, metaphosphates, divinyl compounds, bisaziridines, etc. The crosslinking may also be unipolymeric (one polymer, whether alone or after crosslinking one portion of a mixture of two or more hydrophilic polymers), dipolymeric (both polymers of a mixture of two polymers are crosslinked in the presence of each other), or multipolymeric (all polymers in a mixture of three or more polymers are crosslinked in the presence of each other, or a subset of two or more polymers in a mixture of two or more polymers plus one polymer are crosslinked before mixing with the remaining polymers).

[0049] A mixture of two or more hydrophilic polymers can have equal proportions of each polymer (e.g., as measured by mass, moles, volume, or other measure), or an excess of one or more polymers relative to the other. When a mixture of two polymers is present, the ratio of one polymer to another in the mixture of two polymers can be, for example, about 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, and ratios therebetween. In a mixture of three or more polymers, each can vary in proportion relative to the other polymers in a manner similar to the mixture of two polymers described above. That is, one or more polymers can be in excess of any other polymer, or all polymers can have equal proportions (e.g., by mass, moles, volume, or other measure). For example, a mixture of three polymers (A:B:C) can have ratios of 5:2:1, 1:1:1, 3:10:1, 4:9:1, 1:15:3, 20:1:7, etc.

[0050] In some embodiments, the support matrix is ​​a mixture of cellulose and pullulan, which can have equal proportions of pullulan and cellulose, an excess of pullulan to cellulose, or an excess of cellulose to pullulan. The cellulose to pullulan ratio can be, for example, about 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, and ratios therebetween. In some embodiments, the cellulose to pullulan ratio is 2:1.

[0051] In some embodiments, the indicator layer further comprises an acidic buffer. In some embodiments, the pH is between about pH 1 and 6, pH 1 and 5, pH 2 and 7, pH 2 and 6, pH 3 and 6, pH 4 and 6, pH 2 and 5, pH 3 and 5, pH 2.5 and 5.5, pH 2.5 and 5, pH 2.5 and 4.5, pH 3 and 5.5, pH 3.5 and 5.5, pH 3.5 and 5, or about any one of pH 2.5, pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, or pH 6. In certain embodiments, the pH is between about pH 3.5 and pH 4.5, and in other certain embodiments, the pH of the indicator layer is about pH 4.

[0052] Suitable buffers include, for example, maleate (pKal), phosphate (pKal), glycine (pKal), citrate (pKal), glycylglycine (pKal), malate (pKal), formate, citrate (pKal), succinate (pKal), acetate, propionate, malate (pKal), pyridine, piperazine (pKal), cacodylate, succinate (pKal), MES (2-(N-morpholino)ethanesulfonic acid), phosphate (pKal), citrate (pKal), maleate (pKal), tartrate, and others suitable for supporting biological reactions at acidic pH levels. Appropriate pH buffering behavior is generally observed within + / - 1 unit of the pKal value; therefore, buffers with pKal values ​​less than 7 may be suitable for the indicator layer.

[0053] In some embodiments, the indicator layer further comprises one or more bile salts. As described herein, bile salts include, for example, cholate, taurocholate, and deoxycholate, among others. Sodium and potassium salts are most commonly used, with sodium salts being the most common counterion. In some embodiments, the bile salt comprises sodium cholate, sodium deoxycholate, sodium taurocholate, sodium taurodeoxycholate, sodium deoxytaurocholate, or a mixture of two or more thereof, and in some embodiments, the bile salt is sodium taurodeoxycholate.

[0054] In some embodiments, the indicator layer further comprises one or more surfactants. Suitable surfactants may include those having a carboxyl group, a sulfonic acid group, a sulfate group, or a phosphate group as a hydrophilic group. Anionic surfactants having a sulfonic acid group include alkyl benzene sulfonates (e.g., sodium dodecylbenzene sulfonate; SDBS), alkyl naphthalene sulfonates, alkyl sulfates, polyoxyethylene alkyl ether sulfates, α-olefin sulfonates, and N-acylmethyl taurine salts. The anionic surfactant preferably does not inhibit lipase activity or inactivate any enzymes added to the device described herein.

[0055] In some embodiments, the surfactant is an alkylbenzenesulfonate having an alkyl chain containing 10 to 14 carbon atoms, and in some embodiments, sodium dodecylbenzenesulfonate is used. The sodium salt is the most common salt, but potassium or lithium salts can also be used.

[0056] In some embodiments, the one or more surfactants comprise the nonionic surfactants described above. In some embodiments, the nonionic surfactants according to the present disclosure have a hydrophilic-lipophilic balance (HLB) ranging from about 15 to 18, and in some embodiments, the nonionic surfactant is soluble in polar solvents, such as, but not limited to, alcohols, such as methanol, ethanol, n-propanol, isopropanol, and n-butanol. In some embodiments, the nonionic surfactant is tergitol. TM , e.g., Tergitol TM It is 15S-30.

[0057] In some embodiments, the isolation layer further comprises a polyvinylpyrrolidone support matrix and a non-ionic surfactant having an HLB between 15 and 18 that is also soluble in alcohol solvents.

[0058] In some embodiments, the chromogenic substrate is DGGR, which in some embodiments is emulsified with a bile (acid) salt, such as cholate, in a buffer. Bile salts include, for example, alkali metal salts of cholic acid, taurocholic acid, deoxycholic acid, taurodeoxycholic acid, and glycodeoxycholic acid. Because DGGR is unstable at neutral or basic pH conditions, DGGR may be maintained at an acidic pH of about 3.5 to about 4.5. In some embodiments, the buffer is a tartrate buffer at pH 4.0.

[0059] In some embodiments where the chromogenic substrate is DGGR, DGGR is not emulsified, but rather is a soluble or miscible component in an organic solvent or solvent system, combined with one or more hydrophilic polymers (e.g., polyvinylpyrrolidone (PVP)) described herein that are compatible with the organic solvent or solvent system. Suitable solvents and solvent systems are polar organic solvents, such as ethanol, isopropyl alcohol, methanol, etc.

[0060] In some embodiments, the device also includes a support layer that provides a rigid or semi-rigid physical foundation for the layers of the device. In some embodiments, the support layer is optically transparent and impermeable to aqueous liquids, e.g., having a thickness of 50 μm to 5 mm (and in some embodiments, between about 120 μm and 180 μm), and includes a transparent support in film or sheet form comprising a material, in some embodiments, a polymer such as polyethylene terephthalate (PET), polycarbonate of bisphenol A, polystyrene, cellulose esters (e.g., cellulose diacetate, cellulose triacetate, or cellulose acetate propionate), or other suitable material such as glass. In some embodiments, the support layer is PET.

[0061] In some embodiments, the device also includes a primer layer on the support layer that promotes and / or strengthens bonding of other layers of the device to the support layer. In some embodiments, the primer layer is located between the support layer and the indicator layer, while in other embodiments, the location of certain layers is changed, with the primer layer being located between the support layer and the basic buffer layer. Alternatively or additionally, a physical or chemical activation treatment can be performed on the surface of the support layer to enhance adhesion.

[0062] In some embodiments, the primer layer comprises a hydrogel, such as D4 hydrogel, which is a crosslinked three-dimensional hydrophilic network that has the ability to swell but resist dissolution when placed in water or other biological fluids. In some embodiments, the primer layer also comprises a support matrix such as cellulose and / or cellulose derivatives (e.g., carboxymethylated cellulose, methylated cellulose, hydroxyethylated cellulose, and hydroxypropylated cellulose). In certain embodiments, the primer layer comprises D4 hydrogel and cellulose.

[0063] In another aspect, the present disclosure provides a device that, in some embodiments, also includes a separate, discrete coated propagation layer positioned between the basic buffer layer and the isolation layer.

[0064] For embodiments including a separate propagation layer, the basic buffer layer can further include a support matrix. As described for the isolation layer and indicator layer, the support matrix present in the basic buffer layer in some embodiments including a separate propagation layer includes at least one hydrophilic polymer, and in some embodiments, the support matrix includes a mixture of two or more hydrophilic polymers. The hydrophilic polymer is water-soluble or water-swellable and can be natural, synthetic, or semi-synthetic. Hydrophilic polymers of the present disclosure include starch, pullulan, pullulan derivatives, cellulose, cellulose derivatives (e.g., carboxymethylated cellulose, methylated cellulose, hydroxyethylated cellulose, and hydroxypropylated cellulose), scleroglucan, elsinan, levan, alternants, dextran, agarose, gelatin (e.g., acid-treated gelatin and deionized gelatin), gelatin derivatives (e.g., phthalated gelatin and hydroxyacrylate-grafted gelatin), acrylamide polymers, such as copolymers comprising acrylamide and various vinyl monomers, vinylpyrrolidone polymers (e.g., polyvinylpyrrolidone), and acrylate polymers.

[0065] The hydrophilic polymers of the present disclosure may be non-crosslinked or crosslinked, as described elsewhere herein.

[0066] A mixture of two or more hydrophilic polymers can have equal or unequal proportions, as described elsewhere herein. In some embodiments of this aspect of the present disclosure, the support matrix included in the basic buffer layer is a mixture of cellulose and pullulan, which can have equal proportions of pullulan and cellulose, an excess of pullulan relative to cellulose, or an excess of cellulose relative to pullulan. The ratio of cellulose to pullulan can be, for example, about 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, and ratios therebetween. In some embodiments, the ratio of cellulose to pullulan is 2:1.

[0067] In some embodiments of this aspect of the present disclosure, the transmission layer further comprises one or more components that facilitate sample transmission and absorption. In some embodiments, the one or more components include polymeric binders, viscosity modifiers, plasticizers, biocidal components, lubricants, antifoaming agents, particles ranging in size from 1 to 100 μm, and combinations thereof, as known in the art, e.g., U.S. Pat. No. 3,992,158, U.S. Pat. No. 4,258,001, and U.S. Pat. No. 4,670,381. The relative concentration may be defined as the pigment volume concentration (PVC), as described elsewhere herein.

[0068] In some embodiments of this aspect of the disclosure, the basic buffer layer has a pH between about pH 7 and 9, e.g., using bicine buffer or other basic buffers described herein, and further comprises one or more surfactants and a pullulan-cellulose support matrix (each of the surfactants and support matrices described above and herein), with the proviso that in some embodiments, the basic buffer layer does not comprise carboxymethylcellulose or polyethylene glycol, e.g., PEG 300. In some embodiments of this aspect in which a separate propagation layer is present, the isolation layer does not comprise a surfactant (e.g., SDBS) or a bile salt (e.g., cholate, taurodeoxycholate, etc.).

[0069] In certain embodiments of all aspects of the present disclosure, the basic buffer layer comprises a bicine buffer, one of the one or more surfactants is SDBS, and the pullulan-cellulose support matrix comprises an excess of cellulose relative to pullulan.

[0070] In some embodiments, one or more of the layers of the devices described herein, e.g., the basic buffer layer, the isolation layer, and the indicator layer, are coated and dried on an optically transparent substrate, either directly on the substrate or, in some embodiments, on a primer layer that promotes and / or strengthens bonding of the other layers of the device to the substrate. In some embodiments, each successive layer of the device, from bottom to top, is coated and dried before adding the next layer. For example, in embodiments having an optically transparent substrate and a primer layer, the primer layer is coated and dried, the indicator layer is coated and dried on the primer layer, the isolation layer is coated and dried on the indicator layer, and the basic buffer layer is coated and dried on the isolation layer.

[0071] In some embodiments, the basic buffer layer is between about 150 μm and 250 μm thick, the isolation layer is between about 25 μm and 75 μm thick, the indicator layer is between about 25 μm and 75 μm thick, and the primer layer is between about 5 μm and 20 μm thick. In other embodiments, the basic buffer layer is about 200 μm thick, the isolation layer is about 50 μm thick, the indicator layer is about 45 μm thick, and the primer layer is about 12 μm thick.

[0072] In some embodiments where a separate propagation layer is present, the basic buffer layer is between about 150 μm and 250 μm thick, the propagation layer is between about 100 μm and 200 μm thick, the isolation layer is between about 25 μm and 75 μm thick, the indicator layer is between about 25 μm and 75 μm thick, and the primer layer is between about 5 μm and 20 μm thick. In one particular embodiment, the basic buffer layer is about 200 μm thick, the separate propagation layer is about 150 μm thick, the isolation layer is about 50 μm thick, the indicator layer is about 45 μm thick, and the primer layer is about 12 μm thick.

[0073] In some embodiments, the device includes a set of colorimetric standards for correlating the color change in a mixture containing an animal sample, bile salts, colipase, and a chromogenic substrate to the amount of pancreatic lipase in the sample. The colorimetric standards include a color chart containing multiple colors for correlating the color change in a mixture containing a sample, bile salts, colipase, and a chromogenic substrate to the amount of pancreatic lipase in the sample, the mixture being generated within the device's layer after application of the sample to the top layer of the device. Colorimetric standards can also be generated using a standard curve, for example, by testing multiple known amounts of pancreatic lipase for color development upon cleavage of the glutaric acid-(6-methylresorufin) ester from the DGGR substrate, which spontaneously decomposes in alkaline solution to form glutaric acid and the chromophore methylresorufin. Depending on the animal sample being tested, a standard curve of color versus pancreatic lipase concentration can be generated, ranging from a pancreatic lipase-free standard sample to a standard sample containing high levels of canine or feline pancreatic lipase, from which the pancreatic lipase concentration of the animal sample can be determined.

[0074] In another aspect, a device for detecting the presence of pancreatic lipase in an animal sample includes an additional arrangement of layers. For example, in some embodiments, the device includes, in top-to-bottom order, (i) an indicator layer, (ii) a separator layer, (iii) a basic buffer layer, and (iv) a system for optically determining the amount of color change occurring in a mixture of the sample, bile salts, colipase, and chromogenic substrate. In some embodiments, such a device also includes a primer layer and / or an optically transparent substrate below the basic buffer layer, for example, if the various layers are not themselves supportive or otherwise do not have the desired rigidity.

[0075] In some embodiments of this aspect, the device also includes a separate propagation layer located between the indicator layer and the isolation layer or between the isolation layer and the basic buffer layer. As with other aspects and embodiments described herein that include a separate propagation layer, for embodiments of this aspect, the basic buffer layer can further include a support matrix. In some embodiments that include a separate propagation layer, the support matrix present in the basic buffer layer includes at least one hydrophilic polymer (crosslinked or non-crosslinked), and in some embodiments, the support matrix includes a mixture of two or more hydrophilic polymers as described elsewhere herein.

[0076] The mixture of two or more hydrophilic polymers for embodiments of this aspect can have equal or unequal ratios, as described elsewhere herein. In some embodiments of this aspect of the disclosure, the support matrix included in the basic buffer layer is a mixture of cellulose and pullulan, and in some embodiments, the ratio of cellulose to pullulan is about 2:1.

[0077] In some embodiments of this aspect of the disclosure, the separate propagation layer further comprises one or more components that facilitate sample propagation and absorption. In some embodiments, the one or more components include polymeric binders, viscosity modifiers, plasticizers, biocidal components, lubricants, antifoaming agents, particles ranging in size from 1 to 100 μm, and combinations and concentrations thereof, known in the art, e.g., U.S. Pat. Nos. 3,992,158, 4,258,001, and 4,670,381, and described elsewhere herein.

[0078] In some embodiments of this aspect of the disclosure, the basic buffer layer is dried in a buffer between about pH 7 and 9, including, for example, bicine buffer or other basic buffers described herein, and can further include one or more surfactants and a pullulan-cellulose support matrix (each of the surfactants and support matrices described above and herein), with the proviso that in some embodiments, the basic buffer layer does not include carboxymethylcellulose or polyethylene glycol, such as PEG 300. In some embodiments of this aspect in which a propagation layer is present, the isolation layer does not include a surfactant (e.g., SDBS) or a bile salt (e.g., cholate, taurodeoxycholate, etc.).

[0079] In another aspect, the present disclosure provides a method for detecting the presence or amount of pancreatic lipase in an animal sample, the method comprising: (i) providing a device for detecting the presence or amount of pancreatic lipase in an animal sample, the device comprising: a basic buffer layer; an isolation layer comprising colipase; an indicator layer comprising a chromogenic substrate; and a system for optically determining the amount of color change in a mixture of the sample, the colipase, and the chromogenic substrate; (ii) introducing the sample onto the basic buffer layer (or other top layer described herein) for diffusion through the basic buffer layer, the isolation layer, and the indicator layer (i.e., subsequent layers); (iii) determining the color change in the device; and (iv) correlating the color change with the presence or amount of pancreatic lipase in the sample.

[0080] Essentially, this method is based on the catalytic cleavage of an alkaline lipase solution of the specific chromogenic lipase substrate DGGR, which can be emulsified with bile acids as described in U.S. Pat. No. 4,988,496 (incorporated herein by reference in its entirety) or dissolved in polar solvents such as methanol, ethanol, n-propanol, isopropanol, etc. This substrate is specific for pancreatic lipase in the presence of colipase, which activates pancreatic lipase but not other lipolytic enzymes found in serum. DGGR reagent and colipase are available from Roche Diagnostics GmbH (Mannheim, Germany).

[0081] In some method embodiments, the animal sample is a canine or feline sample, such as serum or blood, and in some embodiments, the chromogenic substrate comprises 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methyl-resorufin) ester (DGGR).

[0082] In some method embodiments, the basic buffer layer comprises a basic buffer, which in some embodiments has a pH between about pH 7-10, pH 7-9, pH 7-8, pH 8-10, pH 8-9, pH 7.5-10, pH 7.5-9.5, pH 7.5-9, pH 7.5-8.5, pH 8-10, pH 8-9.5, pH 8-9, or any one of about pH 7, pH 7.5, pH 8, pH 8.4, pH 9, pH 9.5, or pH 10. In some embodiments, the basic buffer layer is dried in a buffer between about pH 7-9.

[0083] Suitable basic buffers are described herein and include buffers that are suitable for biological reactions and have a pKa value of 7 or greater than 7. In certain embodiments, the basic buffer layer comprises a bicine buffer at pH 8.4.

[0084] In some embodiments, the basic buffer layer may further comprise calcium, which is typically provided as calcium ions in the form of calcium salts including, for example, calcium chloride, calcium carbonate, calcium citrate, calcium lactate, calcium sulfate, calcium malate, and the like.

[0085] In some embodiments, the basic buffer layer further comprises colipase, which may be present at a concentration of about 1 mg / L to about 50 mg / L, e.g., 5 to 45 mg / L, 10 to 30 mg / L, or 15 to 25 mg / L. In some embodiments comprising colipase in the basic buffer layer, the isolation layer does not contain colipase.

[0086] In some embodiments, the basic buffer layer further comprises one or more surfactants. Suitable surfactants are described in detail elsewhere herein. In some embodiments, the surfactant is an alkylbenzenesulfonate having an alkyl chain containing 10 to 14 carbon atoms. In some embodiments, sodium dodecylbenzenesulfonate is used. In some embodiments, one or more nonionic surfactants are used, some of which are soluble in polar solvents such as methanol, ethanol, and isopropanol. Sodium salts are the most common salts. However, potassium or lithium salts can also be used.

[0087] In some embodiments, the basic buffer layer further comprises one or more components that facilitate sample propagation and absorption, as described above and in, for example, U.S. Patent No. 3,992,158, U.S. Patent No. 4,258,001, and U.S. Patent No. 4,670,381. The relative concentration of binder (and, optionally, viscosity modifier, plasticizer, biocide, lubricant, antifoaming agent, etc.) to "pigment" can again be defined in terms of pigment volume concentration (PVC), with suitable PVCs herein ranging from about 87% to 97%, inclusive.

[0088] In some embodiments, the basic buffer layer is in the form of a membrane. For example, a semipermeable membrane (e.g., an IPOC membrane (International Point of Car, Inc., Toronto, Canada)) is coated or impregnated with the components of the basic buffer layer (basic buffer and, optionally, surfactant and / or colipase), dried, laminated to a separator layer, etc. Preferred semipermeable membranes have consistently sized pores and / or a regular porous structure that act to facilitate sample propagation and absorption in addition to providing buffering capacity from the basic buffer components.

[0089] In some embodiments of the present method, the basic buffer layer further comprises a thickener comprising at least one hydrophilic polymer. Hydrophilic polymeric thickeners are described elsewhere herein, but some include cellulose, cellulose derivatives, acrylamide polymers, such as copolymers comprising acrylamide and various vinyl monomers, vinylpyrrolidone polymers (e.g., polyvinylpyrrolidone), and acrylate polymers. In certain embodiments, the basic buffer layer comprises carboxymethylcellulose.

[0090] In some embodiments of the methods described herein, the isolation layer further comprises a support matrix. The support matrix comprises at least one hydrophilic polymer, and in some embodiments, the support matrix comprises a mixture of two or more hydrophilic polymers. Suitable hydrophilic polymers can be natural, synthetic, or semi-synthetic, examples of which are described elsewhere herein. The hydrophilic polymer can be non-crosslinked or crosslinked.

[0091] A mixture of two or more hydrophilic polymers can have equal proportions of each polymer, or an excess of one or more polymers relative to the other, as described elsewhere herein. In some embodiments, the support matrix is ​​a mixture of cellulose and pullulan in a 2:1 ratio, and in some embodiments, the support matrix is ​​polyvinylpyrrolidone.

[0092] In some method embodiments, the isolation layer further comprises an acidic buffer having a composition and pH value or range of values ​​described elsewhere herein. In some embodiments, the pH range is between about pH 3.5 and pH 4.5 to help maintain stability of the DGGR in the indicator. Suitable acidic buffers are described elsewhere herein.

[0093] In some embodiments, one or more layers of a device utilized in the disclosed methods, such as the basic buffer layer and / or the indicator layer, contain one or more bile salts. As described herein, bile salts include, for example, cholate, taurocholate, and deoxycholate, among others. Sodium and potassium salts are most commonly used, with sodium salts being the most common counterion. In some embodiments, the bile salt comprises sodium cholate, sodium deoxycholate, sodium taurocholate, sodium taurodeoxycholate, sodium deoxytaurocholate, or a mixture of two or more thereof; in some embodiments, the bile salt is sodium taurodeoxycholate. In some method embodiments, the isolation layer further comprises one or more bile salts.

[0094] In some method embodiments, the separating layer further comprises one or more surfactants. Suitable surfactants are described elsewhere herein, and some can include alkylbenzene sulfonates having alkyl chains containing 10 to 14 carbon atoms (e.g., sodium dodecylbenzene sulfonate; SDBS). In some embodiments, one or more nonionic surfactants are used, some of which are soluble in polar solvents such as methanol, ethanol, and isopropanol. The surfactant preferably does not inhibit lipase activity or inactivate any enzymes added to the devices described herein.

[0095] In some method embodiments, the indicator layer further comprises a support matrix. The support matrix comprises at least one hydrophilic polymer, and in some embodiments, the support matrix comprises a mixture of two or more hydrophilic polymers. Suitable hydrophilic polymers can be natural, synthetic, or semi-synthetic, and can be uncrosslinked or crosslinked, examples of which are described elsewhere herein.

[0096] A mixture of two or more hydrophilic polymers can have equal proportions of each polymer, or an excess of one or more polymers relative to the other, as described elsewhere herein. In some embodiments, the support matrix is ​​a mixture of cellulose and pullulan in a 2:1 ratio.

[0097] In some method embodiments, the indicator layer further comprises an acidic buffer having a composition and pH value or range described elsewhere herein. In certain embodiments, the pH is between about pH 3.5 and pH 4.5, and in other certain embodiments, the pH of the indicator layer is about pH 4 and the acidic buffer is a tartrate buffer.

[0098] In some embodiments, the indicator layer further comprises one or more surfactants. For the isolation layer, suitable surfactants are described elsewhere herein and may include, in part, alkylbenzene sulfonates having alkyl chains containing 10 to 14 carbon atoms (e.g., sodium dodecylbenzene sulfonate; SDBS). In some embodiments, the one or more surfactants comprise the nonionic surfactants described above. In some embodiments, nonionic surfactants according to the present disclosure have a hydrophilic-lipophilic balance ranging from about 15 to 18, and in some embodiments, the nonionic surfactant is soluble in polar solvents, such as, but not limited to, alcohols, such as methanol, ethanol, n-propanol, isopropanol, and n-butanol. In some embodiments, the nonionic surfactant is tergitol. TM , e.g., Tergitol TM It is 15S-30.

[0099] In some embodiments, the isolation layer further comprises a polyvinylpyrrolidone support matrix and a non-ionic surfactant that is also soluble in alcohol solvents and has an HLB between 15 and 18. The surfactant preferably does not inhibit lipase activity or inactivate any enzymes added to the devices described herein.

[0100] In some method embodiments, the chromogenic substrate in the indicator layer is DGGR, which in some embodiments is emulsified with one or more bile (acid) salts, such as cholate, in a buffer. Suitable bile salts are described elsewhere herein, and include, for example, alkali metal salts of cholic acid, taurocholic acid, desoxycholic acid, taurodesoxycholic acid, glycodesoxycholic acid, and the like.

[0101] In some method embodiments, the chromogenic substrate is DGGR, which is not emulsified, but rather is a soluble or miscible component in an organic solvent or solvent system in combination with one or more hydrophilic polymers (e.g., vinylpyrrolidone polymers such as polyvinylpyrrolidone) described herein that are compatible with the organic solvent or solvent system. Suitable solvents and solvent systems are polar organic solvents, such as ethanol, isopropyl alcohol, methanol, etc.

[0102] In some method embodiments, the device also includes a support layer that provides a rigid or semi-rigid physical foundation for the layers of the device. The support layer is optically transparent, impermeable to aqueous liquids, and of a thickness and composition as described elsewhere. In some embodiments, the support layer is made of PET.

[0103] In some method embodiments, the device also includes a primer layer on the support layer that promotes and / or strengthens bonding of other layers of the device to the support layer. In some embodiments, the primer layer is located between the support layer and the indicator layer, while in other embodiments, the location of certain layers is altered, with the primer layer being located between the support layer and the basic buffer layer. Alternatively or additionally, a physical or chemical activation treatment can be performed on the surface of the support layer to enhance adhesion. As described more fully elsewhere herein, in some embodiments, the primer layer includes a hydrogel, such as D4 hydrogel. In some embodiments, the primer layer also includes a support matrix, such as cellulose and / or a cellulose derivative (e.g., carboxymethylated cellulose, methylated cellulose, hydroxyethylated cellulose, and hydroxypropylated cellulose). In certain embodiments, the primer layer includes D4 hydrogel and cellulose.

[0104] In some embodiments, the chromogenic substrate is emulsified DGGR, the at least one surfactant is SDBS, and at least one of the isolation layer and the indicator layer further comprises a support matrix comprising pullulan-cellulose.

[0105] In some embodiments, the chromogenic substrate is DGGR dissolved in a polar solvent, the at least one surfactant is a non-ionic surfactant having a hydrophobic-lipophilic balance of between about 15 and 18, and at least one of the isolation layer and the indicator layer further comprises a support matrix comprising PVP.

[0106] In another method aspect, the present disclosure provides a method for detecting the presence or amount of pancreatic lipase in an animal sample, wherein the device further comprises a separate transmission layer. In some embodiments, the separate transmission layer is located between the basic buffer layer and the isolation layer.

[0107] For embodiments including a separate propagation layer, the basic buffer layer can further include a support matrix. As described for the isolation layer and indicator layer, in some embodiments including a separate propagation layer, the support matrix in the basic buffer layer, if present, includes at least one hydrophilic polymer or a mixture of two or more hydrophilic polymers, which may be natural, synthetic, or semi-synthetic. Hydrophilic polymers of the present disclosure are described elsewhere herein, but include, among others, pullulan, pullulan derivatives, cellulose, cellulose derivatives (e.g., carboxymethylated cellulose, methylated cellulose, hydroxyethylated cellulose, and hydroxypropylated cellulose), and acrylamide polymers. The hydrophilic polymers of the present disclosure can be non-crosslinked or crosslinked, as described elsewhere herein.

[0108] The mixture of two or more hydrophilic polymers can have equal or unequal proportions, as described elsewhere herein. In some embodiments of this aspect of the disclosure, the support matrix included in the basic buffer layer is a mixture of cellulose and pullulan, which in some embodiments has a 2:1 ratio of cellulose to pullulan.

[0109] In some embodiments of this method aspect of the present disclosure, the separate propagation layer further comprises one or more components that facilitate sample propagation and absorption. In some embodiments, the one or more components include polymeric binders, viscosity modifiers, plasticizers, biocidal components, lubricants, antifoaming agents, particles in the size range of 1 to 100 μm, and combinations thereof, known in the art, e.g., U.S. Pat. No. 3,992,158, U.S. Pat. No. 4,258,001, and U.S. Pat. No. 4,670,381.

[0110] In some embodiments, the basic buffer layer further comprises one or more surfactants. Suitable surfactants are described in detail elsewhere herein. In some embodiments, the surfactant is an alkylbenzenesulfonate having an alkyl chain containing 10 to 14 carbon atoms, and in some embodiments, sodium dodecylbenzenesulfonate is used. Sodium salts are the most common salts; however, potassium or lithium salts can also be used.

[0111] In some embodiments, the basic buffer layer further comprises polyethylene glycol (or a PEG substitute), as described elsewhere herein. In certain embodiments, one or more of PEG200 through PEG600 are included, in other embodiments, one or more of PEG200 through PEG400 are included, in some embodiments, PEG200, PEG300, or PEG400 are included, and in some embodiments, PEG300 is included.

[0112] In some embodiments of this method aspect, the basic buffer layer is dried in a buffer between about pH 7-9, including, for example, bicine buffer or other basic buffers described herein, and further comprises one or more surfactants and a pullulan-cellulose support matrix (each of the surfactants and support matrices described above and herein), with the proviso that in some embodiments, the basic buffer layer does not comprise carboxymethylcellulose or polyethylene glycol, such as PEG 300. In some embodiments of this method aspect where a separate propagation layer is present, the isolation layer does not comprise a surfactant (e.g., SDBS) or a bile salt (e.g., cholate, taurodeoxycholate, etc.).

[0113] In certain embodiments of all aspects of the present disclosure, the basic buffer layer comprises a bicine buffer, one of the one or more surfactants is SDBS, and the pullulan-cellulose support matrix comprises an excess of cellulose relative to pullulan.

[0114] In embodiments of this aspect, the basic buffer layer, the isolation layer, and the indicator layer have layer thicknesses as described elsewhere herein, whether or not separate transmissions are present.

[0115] In some embodiments of this aspect, the device includes a set of colorimetric standards for relating a color change in a mixture comprising an animal sample, bile salts, colipase, and a chromogenic substrate to the amount of pancreatic lipase in the sample, as described in more detail elsewhere herein.

[0116] In another aspect, the present disclosure provides methods for detecting the presence or amount of pancreatic lipase in an animal sample, wherein the device comprises an alternative arrangement of layers. For example, in some embodiments, the device comprises (i) an indicator layer, (ii) a separator layer, (iii) a basic buffer layer, and (iv) a system for optically determining the amount of color change occurring in a mixture of the sample, bile salts, colipase, and chromogenic substrate. In some embodiments, such devices also include a primer layer below the basic buffer layer, and / or an optically transparent substrate, for example, if the various layers are not themselves supportive or otherwise do not have the desired rigidity.

[0117] In some embodiments of this method aspect, the device includes a separate propagation layer located between the indicator layer and the isolation layer or between the isolation layer and the basic buffer layer. As with other aspects and embodiments described herein that include a separate propagation layer, for embodiments of this aspect, the basic buffer layer can further include a support matrix. The support matrix in the basic buffer layer in embodiments that include a separate propagation layer, if present, includes at least one hydrophilic polymer (crosslinked or non-crosslinked); in some embodiments, the support matrix includes a mixture of two or more hydrophilic polymers, as described elsewhere herein. Such mixtures of two or more hydrophilic polymers can have equal or unequal proportions, in ratios described elsewhere herein. In some embodiments of this aspect of the disclosure, the support matrix included in the basic buffer layer is a mixture of cellulose and pullulan; in some embodiments, the ratio of cellulose to pullulan is about 2:1.

[0118] In some embodiments of this aspect of the present disclosure, the separate propagation layer further comprises one or more components that facilitate sample propagation and absorption. In some embodiments, the one or more components include polymeric binders, viscosity modifiers, plasticizers, biocidal components, lubricants, antifoaming agents, particles in the size range of 1 to 100 μm, and combinations thereof, known in the art, e.g., U.S. Pat. No. 3,992,158, U.S. Pat. No. 4,258,001, and U.S. Pat. No. 4,670,381.

[0119] In some embodiments of this method aspect, the basic buffer layer is dried in a buffer between about pH 7-9, including, for example, bicine buffer or other basic buffers described herein, and further comprises one or more surfactants and a pullulan-cellulose support matrix (each of the surfactants and support matrices described above and herein), with the proviso that in some embodiments, the basic buffer layer does not comprise carboxymethylcellulose or polyethylene glycol, such as PEG 300. In some embodiments of this method aspect where a separate propagation layer is present, the isolation layer does not comprise a surfactant (e.g., SDBS) or a bile salt (e.g., cholate, taurodeoxycholate, etc.).

[0120] In all embodiments of the present disclosure, canine or feline pancreatic lipase can be detected in blood samples, including whole blood, plasma, or serum. For example, if the sample is a serum sample, the sample may comprise a fraction, an aliquot, a drop, a portion, or a volume of serum collected as whole blood from a patient source using standard sampling tubes, such as Li-, Na-, or NH4-heparinized plasma, collected in a standard manner. Blood samples may be centrifuged before performing the methods of the present invention. EDTA, oxalate, fluoride, or citrated plasma inhibit lipase activity and are therefore less desirable for storing test blood samples. Test samples may be collected from animal sources using techniques known to those skilled in the art, including, but not limited to, those described or referenced in "Manual of Clinical Microbiology" (6th ed.) 1995, edited by P.R. Murray, E.J. Baron, M.A.P. Faller, F.C. Tenover, and R.H. Olken.

[0121] An animal sample is applied to the top layer of a device for detecting the presence or amount of pancreatic lipase in an animal sample. The sample typically has a volume of between about 1 μL and 25 μL, or between about 2 μL and 20 μL, or between about 3 μL and 15 μL, or between about 4 μL and 12 μL, or between about 5 μL and 10 μL, or between about 6 μL and 8 μL, or between about 5 μL and 20 μL, or between about 1 μL and 12 μL, or between about 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, or about 25 μL.

[0122] After an animal sample is applied to the top layer of the device, if pancreatic lipase is present in the sample, color development occurs over time. Color development generally occurs over approximately 5-15 minutes, at which point the color change can be determined by human or machine-based (e.g., spectrophotometric) observation. The observed color change is then compared to a standard as described elsewhere herein and correlated with the presence or amount of pancreatic lipase.

[0123] In yet another aspect, the present disclosure provides a kit for carrying out the method, comprising a device for detecting the presence or amount of pancreatic lipase in an animal sample and instructions for use of the device. The kit components may be enclosed in an additional sealed container for transport and storage, such as a foil pouch, and may contain a desiccant. The kit may also include specimen collection tubes for serum collection, such as Li-, Na-, or NH4-heparin tubes.

[0124] For all aspects of the present disclosure, one or more coating techniques can be used to create the various layers of the device for detecting the presence or amount of pancreatic lipase in an animal sample. For example, knife-over-air coating (floating knife), knife-over-roll coating, roll coating, dipping, roll-to-roll coating (e.g., Dynacoat® from Frontier), TM, Tonda, PA), web coating, kiss coating, gravure coating, metering rod or Myer bar coating, comma direct coating, comma reverse coating, reverse roll coating, slot orifice coating, calendaring, immersion or dip coating, and curtain coating may be used for the basic buffer layer, isolation layer, indication layer, and, where applicable, separate propagation and primer layers.

[0125] For example, for embodiments involving optically transparent substrates, knife-over-air coating can be used to apply each layer sequentially. The knife-over-air coating process involves a knife or blade positioned vertically above the substrate, supported on both sides by two rollers. The coating weight using this technique is derived from the distance between the two rollers and the tension in the substrate. The blade can be raised and lowered to apply additional tension and affect the coating weight and level of penetration (assuming any significant substrate permeability), which affects adhesion and handling. Knife-over-air coating involves a blade positioned above the substrate, which is supported by a lower roller. The blade can be raised or lowered to achieve different coating thicknesses, and the blade shape can be changed to complement the viscosity and rheology of the coating medium.

[0126] The layers may be coated sequentially, from bottom to top, hi some embodiments, at least one layer is dried for a time sufficient to prevent or minimize intermixing with subsequently coated layers.

[0127] For all aspects of the present disclosure, some embodiments can include a filtration layer that can remove unwanted particulates from the sample. For example, the filtration layer can remove red blood cells to prevent them from interfering with the determination of color development in a reagent layer, such as an indicator layer. [Example]

[0128] Example 1 - Preparation and Deposition of Primer Layer 10 g / kg of cellulose was added to 990 g / kg of a 10 wt% HydroMed D4 solution (AdvanSource) in a mixing vessel. The mixture was stirred to disperse the cellulose. Manual shaking was sufficient for dispersion before rolling in a cylindrical vessel at approximately 7.25 cm / sec for at least 12 hours at room temperature. The hydrogel-cellulose mixture was then added to a Dynacoater® (Frontier) and coated onto an optically clear substrate, in this case Melinex® (DuPont Teijin Films). TM ) was coated onto the coated film. Fluid circulation while the coater was running maintained sufficient cellulose dispersion. In this example, the shim width was 6 inches and the thickness was 0.007 inches. The web gap was 150 μm, the web speed was 3 ft / min, the unwind force was 5 lbs, and the unwind torque was 20%. The dryer profile was 110×8 with 0.3 inches of vacuum (i.e., all eight dryers were set at 110°F). The fluid delivery rate was 11.76 mL / min, resulting in a derived wet thickness of 84 μm and a measured dry thickness of 7.5 μm. Those skilled in the art will understand that different coating techniques can be used to achieve similar results and that application parameters can be modified to fine-tune layer characteristics, such as thickness.

[0129] Example 2 - Preparation and Deposition of Indicator Layer I. DGGR High Shear Emulsification Procedure (20-30 mL scale)

[0130] 22.75 grams of DGGR buffer (9.3 mM tartrate, pH 4.0 at room temperature, 0.1 mM calcium chloride, 2.6 g / kg SDBS and 2.4 g / kg sodium taurodeoxycholate) was added to a tared mixing vessel (here a 50 mL conical vial).

[0131] The mixing vessel was placed vertically in an ice bath and secured in place to prevent movement while undergoing shear. The ice bath and conical were placed under a shear mixer (e.g., a Silverson, Rotosolver, or Scott Turbon mixer) with the mixing head lowered so that the liquid level was near the 38 mL mark (if using a conical vial).

[0132] The DGGR / 1-propanol stock solution (25 g / L DGGR and the remainder 1-propanol) was placed on a balance and weighed. Using a needle and syringe, 1.85 grams of the DGGR / 1-propanol solution was aspirated. Over a period of 1-2 minutes, the DGGR concentrate was slowly added to the DGGR buffer solution in the mixing vessel at a rate of approximately 2 mL / min while slowly increasing the mixing speed (e.g., to 6500 RPM on a Silverson mixer). The DGGR solution was mixed on the Silverson at 6500 RPM for 30 minutes.

[0133] After 30 minutes, mixing was stopped, the mixer head was raised, and the liquid in the assembly was allowed to drain into the mixing vessel for 1-2 minutes to maximize recovery. The Silverson was cleaned with 70% IPA by inserting the mix head into an empty 50 mL conical vial, adding 25-30 mL of IPA to the conical vial, immersing the mix head, and mixing at 3000 RPM for 20 seconds. The IPA cleaning solution was combined with the sheared DGGR and stored in a refrigerator at 4-8°C.

[0134] II. DGGR Emulsion Quality Control

[0135] Prior to use in slide layers (e.g., indicator layers), the DGGR emulsion was analyzed for various critical to quality (CTQ) metrics. Dynamic light scattering was used as follows: The emulsion was diluted to 0.1805 g / L using a buffer solution containing 3.75 g / L potassium tartrate and 0.2 g / L sodium dodecylbenzenesulfonate (SDBS), adjusted to pH 4.0. One mL of the diluted emulsion was dispensed into a cuvette, ensuring no bubbles were present, and the cuvette was inserted into a DLS instrument (Zetasizer®, Malvern Panalytical). Replicate samples were scanned to determine the mean particle size and relative polydispersity of the samples. Exemplary results can be seen in Figures 2A and 2B, where the mean particle sizes were 138 nm, 136 nm, and 146 nm for three separate runs, with a size range of approximately 70 nm to approximately 250 nm (Figure 2A), and the correlation function graph confirms this by showing low polydispersity with steep traces and near-perfect overlap for the three runs (Figure 2B). The numerical results are shown in Table 1. [Table 1]

[0136] III. Preparation of the indicator layer

[0137] The 2.27 g / L emulsified DGGR solution was combined with 50 mL of 9.3 mM tartrate buffer (pH 4.0), 3.15 g / L sodium taurodeoxycholate, and 9.0% 1-propanol by volume and mixed in an overhead mixer using a square-pitch impeller at 250 RPM.

[0138] To the DGGR emulsion, 68.0 g / kg pullulan and 137.0 g / kg cellulose were added while mixing with an overhead mixer equipped with a square pitch impeller at 250 RPM. Immediately after adding the pullulan and cellulose, the mixing speed was increased to 350 RPM and mixed for 1 hour. The mixing speed was reduced to 250 RPM and 19.5 mL / kg of a 10 wt% SDBS solution and 0.5 mL / kg of Proclin 300 were added, followed by an additional 10 minutes of mixing.

[0139] The indicator layer was then thoroughly mixed by rotating it in a cylindrical container at 15 RPM (approximately 7.25 cm / sec) for at least 12 hours. The sample was degassed for 10 minutes at -28 inches of mercury vacuum before being added to the Dynacoater.

[0140] In this example, the Dynacoater shim width was 6 inches with a shim thickness of 0.010 inches. The web gap was 250 μm, the web speed was 2.0 ft / min, the unwind force was 5 lbs, and the unwind torque was 20%. The dryer profile was 110×1 and 98×7 (i.e., one dryer set at 110°F and seven dryers set at 98°F) with a 0.3 inch vacuum. The fluid delivery rate was 11.1 mL / min, resulting in a derived wet thickness of 119 μm and a measured dry thickness of 41.3 μm. The resulting coating layer appeared homogeneous and uniform. Example 3 - Preparation and deposition of isolation layer

[0141] To a 1.5 mM tartrate buffer solution (pH 4.0), 2.44 g / kg sodium taurodeoxycholate, 0.5 mL / kg Proclin 300, 0.65 g / kg sodium dodecylbenzenesulfonate (SDBS), and 0.040 g / kg colipase (Roche) were added using a stir plate and stir bar. 68.0 g / kg pullulan and 137.0 g / kg cellulose were slowly added while stirring (200 RPM) under an overhead mixer using a square-pitch impeller. Immediately after the addition of pullulan and cellulose, the mixing speed was increased to 350 RPM and the mixture was mixed for 1 hour.

[0142] The separator layer was then thoroughly mixed by rotating it in a cylindrical container at 15 RPM (approximately 7.25 cm / sec) for at least 12 hours. The sample was degassed at -28 inches of mercury vacuum for 10 minutes before being added to the Dynacoater.

[0143] In this example, the Dynacoater shim width was 6 inches with a shim thickness of 0.010 inches. The web gap was 250 μm, the web speed was 2.0 ft / min, the unwind force was 5 lbs, and the unwind torque was 20%. The dryer profile was 110×1 and 98×7 (i.e., one dryer set at 110°F and seven dryers set at 98°F) with a 0.3 inch vacuum. The fluid delivery rate was 12.2 mL / min, resulting in a derived wet thickness of 131 μm. The resulting coating layer appeared homogeneous and uniform.

[0144] Example 4 - Preparation and Deposition of Basic Buffer Layer In a cylindrical container, add 232.0 g / kg of 100 mM bicine buffer solution (adjusted to pH 8.0 + / - 0.1 with sodium hydroxide pellets or solution), 342.4 g / kg of 1 wt% carboxymethylcellulose (CMC) solution (10 g / kg sodium carboxymethylcellulose powder; remainder deionized (DI) water), 3.7 g / kg polyethylene glycol M to a total volume of 500 mL. nThe base buffer solution was mixed with PEG-300 (i.e., PEG-300 or PEG) and DI water. The base buffer solution was mixed for 10 minutes using a low (shallow) pitch impeller driven by an overhead mixer at 200 RPM. For the 500 mL run, a 2.25 inch, 5-blade impeller was used.

[0145] Mix for approximately 40 minutes to achieve a pigment volume concentration of approximately 93%. 3 A particulate material with a median volume of 1000 mg / L was slowly added to the basic buffer mixture and mixed at 500 RPM for an additional 20 minutes. Then, 53.2 g / kg of Hycar Latex HY26652A (Lubrizol) was added, and the mixture was stirred at 500 RPM for 5 minutes. 5.2 g / kg of sodium taurodeoxycholate was added, followed by 36.8 g / kg of a 10 wt% SDBS solution (100 g / kg sodium dodecylbenzenesulfonate powder, the remainder DI water), followed by stirring at 100 RPM for approximately 2 minutes and then 200 RPM for 5 minutes. The pH of the solution was checked and adjusted as necessary to maintain a pH of 8.0. After each pH adjustment, the mixing vessel was sealed and rotated in a cylindrical container at approximately 7.25 cm / s for at least 20 minutes, after which the pH was rechecked. The pH-corrected sample was then thoroughly mixed by rotating in a cylindrical container at approximately 7.25 cm / s for at least 12 hours. The sample was degassed at -28 inches of mercury vacuum for 10 minutes and then added to a Dynacoater® (Frontier) and coated onto an optically clear substrate, in this case Melinex® (DucPont Teijin Films) TM). Fluid circulation while the coater was running was sufficient to maintain solution homogeneity. In this example, the shim width was 6 inches and the thickness was 0.025 inches. The web gap was 130 μm, the web speed was 2.5 ft / min, the unwind force was 5 lbs, and the unwind torque was 20%. The dryer profile was 115×8 (i.e., all eight dryers were set at 115°F) with no vacuum. The fluid delivery rate was 21.8 mL / min, resulting in a derived wet thickness of 188 μm and a measured dry thickness of 151 μm. The resulting coating layer appeared homogeneous and uniform.

[0146] Parallel samples identical to those described above but lacking deoxycholate were also prepared. The change in viscosity under increasing shear conditions was measured for samples with and without deoxycholate on the day of sample preparation and the day of sample coating. Samples with deoxycholate exhibited a more consistent viscosity on the day of preparation compared to the day of coating, whereas samples without deoxycholate exhibited a significant change in viscosity on the day of sample coating compared to the day of sample preparation.

[0147] Example 5 - Stability To test the storage stability of the dried slides herein, the feline pancreatic lipase concentrations of standardized samples were detected using dried slides frozen and stored at -20°C for 0 days, 15 days, 1 month, 3 months, and 6 months (Figure 4). No statistically significant changes in calculated concentrations were observed over 6 months of storage.

[0148] Example 6 - Sensitivity, Precision Dose-response curves for the detection of feline and canine pancreatic lipase were generated and each tested separately on the same dried slide according to the present invention. Both showed excellent sensitivity and precision, and the canine-specific test (IDEXX Spec cPL;R 2 =0.985; Figure 3A) and a feline-specific test (IDEXX Spec fPL; R 2 = 0.971; Figure 3B).

[0149] Responses from the analyzer using the fully coated dry slide format show good separation and correlation with Spec cPL / fPL.

[0150] Those skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The disclosure set forth herein presently represents preferred embodiments and is exemplary and is not intended as a limitation on the scope of the disclosure. Modifications therein and other uses will occur to those skilled in the art that are encompassed within the spirit of the disclosure as defined by the scope of the claims. Furthermore, the section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0151] No admission is made that any reference, including any non-patent or patent literature, cited herein constitutes prior art. In particular, unless otherwise stated, it is understood that reference to any document herein does not constitute an admission that any of these documents form part of the common general knowledge in the art in the United States or any other country. Any discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are incorporated by reference in their entirety unless expressly indicated otherwise.

[0152] In the event of any discrepancy with any definitions and / or descriptions found in the cited references, the present disclosure shall control.

Claims

1. 1. A device for detecting the presence or amount of pancreatic lipase in an animal sample, comprising: (a) a basic buffer layer; (b) a separator layer comprising colipase; and (c) an indicator layer containing a chromogenic substrate; An apparatus comprising:

2. 10. The apparatus of claim 1, further comprising a system for optically determining the amount of color change occurring in the mixture of the sample, the colipase, and the chromogenic substrate.

3. The device of claim 1 , wherein the animal is a dog or a cat.

4. The device of claim 1 , wherein the animal sample comprises whole blood, serum, or plasma.

5. The device of claim 1 , wherein the animal sample comprises serum.

6. 2. The device of claim 1, wherein the chromogenic substrate comprises 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methyl-resorufin) ester (DGGR).

7. The device of claim 6 , wherein the DGGR is emulsified.

8. The device of claim 6 , wherein the DGGR is dissolved in a polar solvent.

9. The device of claim 8 , wherein the polar solvent is an alcohol.

10. The device of claim 1 , wherein the basic buffer layer has a pH between about 7 and 9.

11. The device of claim 1 , wherein the basic buffer layer further comprises one or more surfactants.

12. 12. The device of claim 11, wherein the surfactant is sodium dodecylbenzene sulfonate (SDBS).

13. 12. The device of claim 11, wherein the surfactant is a non-ionic surfactant having a hydrophilic lipophilic balance (HLB) of between about 15-18.

14. The device of claim 13 , wherein the surfactant is soluble in an alcohol solvent.

15. The device of claim 13, wherein the surfactant is Tergitol 15S-30.

16. The device of claim 1 , wherein the basic buffer layer comprises a bicine buffer.

17. The device of claim 1 , wherein the basic buffer layer further comprises colipase.

18. 10. The device of claim 1, wherein the basic buffer layer is disposed in a semipermeable membrane.

19. 20. The device of claim 18, wherein the basic buffer layer is coated and dried onto the semipermeable membrane or impregnated into the semipermeable membrane.

20. 20. The device of claim 18, wherein the semi-permeable membrane is attached to the separator layer.

21. 10. The device of claim 1, wherein at least one of the isolating layer and the indicator layer further comprises a support matrix.

22. 22. The device of claim 21, wherein the support matrix comprises pullulan-cellulose.

23. 23. The device of claim 22, wherein the pullulan-cellulose support matrix comprises an excess of cellulose relative to pullulan.

24. 24. The device of claim 23, wherein the pullulan-cellulose support matrix comprises a ratio of cellulose to pullulan of about 2:

1.

25. 22. The device of claim 21, wherein the support matrix comprises polyvinylpyrrolidone (PVP).

26. 26. The device of claim 25, wherein at least one of the isolating layer and the indicator layer further comprises a surfactant.

27. 27. The device of claim 26, wherein the surfactant is a non-ionic surfactant having a hydrophilic lipophilic balance (HLB) of between about 15-18.

28. 28. The device of claim 27, wherein the surfactant is soluble in an alcohol solvent.

30. 29. The device of claim 28, wherein the surfactant is Tergitol 15S-30.

31. 22. The device of claim 21, wherein the isolation layer further comprises a polyvinylpyrrolidone support matrix and a non-ionic surfactant having a hydrophilic lipophilic balance (HLB) of between about 15 and 18 and soluble in alcohol solvents.

32. 10. The device of claim 1, wherein the isolating layer and the indicator layer each independently further comprise an acidic buffer.

33. 33. The device of claim 32, wherein the acidic buffer has a pH between about 3.0 and 5.

0.

34. 34. The device of claim 33, wherein the acidic buffer is a tartrate buffer.

35. 10. The device of claim 1, wherein at least one of the separating layer and the indicator layer each independently further comprises one or more bile salts.

36. 36. The device of claim 35, wherein the bile salt is cholate.

37. 37. The device of claim 36, wherein the bile salt is taurodeoxycholate.

38. 38. The device of claim 37, wherein the bile salt is sodium taurodeoxycholate.

39. 10. The device of claim 1, wherein at least one of the isolating layer and the indicator layer each independently further comprises one or more surfactants.

40. 40. The device of claim 39, wherein the surfactant is SDBS.

41. 40. The device of claim 39, wherein the surfactant is a non-ionic surfactant having a hydrophilic lipophilic balance (HLB) of between about 15-18.

42. 42. The device of claim 41, wherein the surfactant is soluble in an alcohol solvent.

43. 43. The device of claim 42, wherein the surfactant is Tergitol 15S-30.

44. The device of claim 1 further comprising a primer layer below the indicator layer.

45. 45. The device of claim 44, wherein the primer layer comprises a hydrogel and a support matrix.

46. 46. ​​The device of claim 45, wherein the support matrix comprises cellulose.

47. The device of claim 1 further comprising an optically transparent substrate beneath the indicator layer.

48. 45. The device of claim 44, further comprising an optically transparent substrate beneath the primer layer.

49. 48. The device of claim 47, wherein the optically transparent substrate comprises polyethylene terephthalate (PET).

50. 49. The device of claim 48, wherein the optically transparent substrate comprises polyethylene terephthalate (PET).

51. 48. The device of claim 47, wherein one or more of the basic buffer layer, the isolation layer, and the indicator layer are coated and dried on the optically transparent substrate.

52. 49. The device of claim 48, wherein one or more of the basic buffer layer, the isolation layer, and the indicator layer are coated and dried onto the primer layer on the optically transparent substrate.

53. 10. The device of claim 1, wherein the basic buffer layer further comprises one or more bile salts and calcium.

54. 10. The device of claim 1, wherein the basic buffer layer is between about 150 μm and 250 μm thick, the isolation layer is between about 25 μm and 75 μm thick, the indicator layer is between about 25 μm and 75 μm thick, and the primer layer is between about 5 μm and 20 μm thick.

55. 55. The device of claim 54, wherein the basic buffer layer is about 200 μm thick, the isolation layer is about 50 μm thick, the indicator layer is about 45 μm thick, and the primer layer is about 12 μm thick.

56. 10. The apparatus of claim 1, wherein the system comprises a set of colorimetric standards for relating a color change in a mixture comprising the sample, the bile salts, the colipase, and the chromogenic substrate to the amount of pancreatic lipase in the sample.

57. 10. The apparatus of claim 1, wherein the system comprises a color chart comprising a plurality of colors for correlating a color change in a mixture comprising the sample, the bile salts, the colipase, and the chromogenic substrate to the amount of pancreatic lipase in the sample.

58. 10. The device of claim 1, wherein the system for determining the amount of color change that occurs comprises a set of standard concentrations of pancreatic lipase.

59. 1. A method for detecting the presence or amount of pancreatic lipase in an animal sample, comprising: i. (a) a basic buffer layer; (b) a separator layer containing colipase; (c) an indicator layer comprising a chromogenic substrate; and (d) a system for optically determining the amount of color change generated in the mixture of the sample, the colipase, and the chromogenic substrate. providing an apparatus comprising: ii. introducing the sample onto the basic buffer layer for diffusion through the basic buffer layer, the isolation layer, and the indicator layer; and iii. Determining a color change in the device; and iv. Correlating the color change with the presence or amount of pancreatic lipase in the sample. A method comprising:

60. 60. The method of claim 59, wherein the animal is a dog or a cat.

61. 60. The method of claim 59, wherein the animal sample comprises whole blood, serum, or plasma.

62. 62. The method of claim 61 , wherein the animal sample comprises serum.

63. 60. The method of claim 59, wherein the chromogenic substrate comprises 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methyl-resorufin) ester (DGGR).

64. 64. The method of claim 63, wherein the DGGR is emulsified.

65. 64. The method of claim 63, wherein the DGGR is dissolved in a polar solvent.

66. 66. The method of claim 65, wherein the polar solvent is an alcohol.

67. 60. The method of claim 59, wherein the basic buffer layer has a pH between about 7 and 9.

68. 60. The method of claim 59, wherein the basic buffer layer further comprises one or more surfactants.

69. 69. The method of claim 68, wherein the surfactant is sodium dodecylbenzene sulfonate (SDBS).

70. 69. The method of claim 68, wherein the surfactant is a non-ionic surfactant having a hydrophilic lipophilic balance (HLB) of between about 15 and 18.

71. 71. The method of claim 70, wherein the surfactant is soluble in an alcohol solvent.

72. 71. The method of claim 70, wherein the surfactant is Tergitol 15S-30.

73. 60. The method of claim 59, wherein the basic buffer layer comprises a bicine buffer.

74. 60. The method of claim 59, wherein the basic buffer layer further comprises colipase.

75. 60. The method of claim 59, wherein the basic buffer layer is disposed in a semipermeable membrane.

76. 76. The method of claim 75, wherein the basic buffer layer is coated and dried onto the semipermeable membrane or impregnated into the semipermeable membrane.

77. 76. The method of claim 75, wherein the semi-permeable membrane is attached to the separator layer.

78. 60. The method of claim 59, wherein at least one of the isolating layer and the indicator layer further comprises a support matrix.

79. 79. The method of claim 78, wherein the support matrix comprises pullulan-cellulose.

80. 80. The method of claim 79, wherein the pullulan-cellulose support matrix comprises an excess of cellulose relative to pullulan.

81. 81. The method of claim 80, wherein the pullulan-cellulose support matrix comprises a ratio of cellulose to pullulan of about 2:

1.

82. 79. The method of claim 78, wherein the support matrix comprises polyvinylpyrrolidone (PVP).

83. 83. The method of claim 82, wherein at least one of the isolating layer and the indicator layer further comprises a surfactant.

84. 84. The method of claim 83, wherein the surfactant is a non-ionic surfactant having a hydrophilic lipophilic balance (HLB) of between about 15 and 18.

85. 85. The method of claim 84, wherein the surfactant is soluble in an alcohol solvent.

86. 86. The method of claim 85, wherein the surfactant is Tergitol 15S-30.

87. 79. The method of claim 78, wherein the isolation layer further comprises a polyvinylpyrrolidone support matrix and a non-ionic surfactant having a hydrophilic lipophilic balance (HLB) of between about 15 and 18 and soluble in alcohol solvents.

88. 60. The method of claim 59, wherein at least one of the isolating layer and the indicator layer further comprises an acidic buffer.

89. 89. The method of claim 88, wherein the acidic buffer has a pH between about 3.0 and 5.

0.

90. 90. The method of claim 89, wherein the acidic buffer is a tartrate buffer.

91. 60. The method of claim 59, wherein at least one of the isolating layer and the indicator layer further comprises one or more bile salts.

92. 92. The method of claim 91, wherein the bile salt is cholate.

93. 93. The method of claim 92, wherein the bile salt is taurodeoxycholate.

94. 94. The method of claim 93, wherein the bile salt is sodium taurodeoxycholate.

95. 60. The method of claim 59, wherein at least one of the isolating layer and the indicator layer further comprises one or more surfactants.

96. 96. The method of claim 95, wherein the surfactant is SDBS.

97. 96. The method of claim 95, wherein the surfactant is a non-ionic surfactant having a hydrophilic lipophilic balance (HLB) of between about 15 and 18.

98. 98. The method of claim 97, wherein the surfactant is soluble in an alcohol solvent.

99. 99. The method of claim 98, wherein the surfactant is Tergitol 15S-30.

100. 60. The method of claim 59, further comprising a primer layer beneath the indicator layer.

101. 101. The method of claim 100, wherein the primer layer comprises a hydrogel and a support matrix.

102. 102. The method of claim 101, wherein the support matrix comprises cellulose.

103. 60. The method of claim 59, further comprising an optically transparent substrate beneath the indicator layer.

104. 101. The method of claim 100, further comprising an optically transparent substrate beneath the primer layer.

105. 104. The method of claim 103, wherein the optically transparent substrate comprises polyethylene terephthalate (PET).

106. 105. The method of claim 104, wherein the optically transparent substrate comprises polyethylene terephthalate (PET).

107. 104. The method of claim 103, wherein one or more of the basic buffer layer, the isolation layer, and the indicator layer are coated and dried on the optically transparent substrate.

108. 105. The method of claim 104, wherein one or more of the basic buffer layer, the isolation layer, and the indicator layer are coated onto the primer layer on the optically transparent substrate and dried.

109. 60. The method of claim 59, wherein the basic buffer layer further comprises one or more bile salts and calcium.

110. 60. The method of claim 59, further comprising an optically transparent substrate beneath the primer layer.

111. 111. The method of claim 110, wherein the optically transparent substrate comprises polyethylene terephthalate (PET).

112. 60. The method of claim 59, wherein the chromogenic substrate is emulsified DGGR, the at least one surfactant is SDBS, and at least one of the isolating layer and the indicator layer further comprises a support matrix comprising pullulan-cellulose.

113. 60. The method of claim 59, wherein the chromogenic substrate is DGGR dissolved in a polar solvent, the at least one surfactant is a non-ionic surfactant having a hydrophilic lipophilic balance (HLB) of between about 15 and 18, and at least one of the isolating layer and the indicator layer further comprises a support matrix comprising PVP.

114. A kit comprising the device of claim 1 and instructions for use of said device.

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