A novel dry stick device construction and method for determining an analyte in a sample using said dry stick device

[0001] The present invention relates to the field of dry strip devices and their use in analyte analysis in a sample. In particular, the present invention relates to an improved construction of a dry strip instrument for the determination of an analyte in a sample, which in particular includes storage stability and performance of the dry strip device.

BACKGROUND ART [0002] There is a growing demand for fast and reliable diagnostic tests in clinical trials. Currently, a significant proportion of diagnostic tests used in clinical trials consist of indicator papers impregnated with several compounds, thus forming an undifferentiated reagent paper. When using such indicator papers, the reagent paper only contacts the material (e.g. body fluid) thus causing a change in color or a change in the intensity of the color, which is used to check whether or not a given effect has been achieved or to quantify the amount of analyte present in the sample.

Currently, clinical laboratory methods used in the diagnosis of physiological status and nutrition, such as diagnosis of mammary gland inflammation in animals, is mainly based on SCC (somatic cell count) e.g. in milk and the diversity of bacterial pathogens in milk samples.

[0003] Thus, traditionally used tests for the presence of inflammation caused by mastitis in animals, until now consisted of rather complicated liquid systems whose tubes, measuring devices, ultraviolet light, apparatus standardization, correction factors depending on temperature increased the frequency of false readings.

ΕΡ 1 982 182 Β1 [0004] Inflammation of the mammary gland affects the integrity of the structure of the mammary gland and at the same time damages the secretory epithelium and the blood-milk barrier. As a result, mastitis affects many components of the milk. The main ingredients like fat, protein and lactose are reduced and the number of enzymes changes.

[0005] Other factors besides those determining SCC that can be used as appropriate indicators for inflammation caused by mastitis in animals are lactate dehydrogenase (LDH) and / or N-acetylglucosaminidase. N-acetyl ^ -D-glucosaminidase, also called N-acetyl glucosaminidase (NAGase), has been recognized as one of the best markers of mastitis. In addition, it has been shown that other enzymes in milk such as LDH can have a similar value as this enzyme as well as NAGase can be used as an appropriate indicator of mastitis.

[0006] According to the state of the art, many methods have been developed for analyzing analytes in liquid samples. These methods can be broadly classified into two types of systems, namely a reaction system in which the reaction is carried out in solution (so-called wet chemistry) and a reaction system in which the reaction is carried out in a solid phase carrier (so-called dry chemistry).

[0007] The wet chemistry analytical reaction involves numerous procedures that differ significantly from the analytical procedure, the so-called manual method, in which no machine is used to use automatic analytical apparatus.

[0008] However, such wet chemistry is mainly carried out in the form of an aqueous solution and requires certain steps that involve various problems. One of the problems that may arise is related to the increased amount of water, which can increase energy consumption. In addition, these activities may require great skill in conducting the analysis, which requires a huge amount of time and work, and waste lyes from production can cause environmental pollution and therefore require further treatment.

[0009] On the other hand, analytical processes using dry chemistry in analytical reactions are also widely used and widely practiced in the form of undifferentiated filter paper and other porous materials

ΕΡ 1 982 182 Β1 impregnated in one or more reagents capable of generating a detectable signal.

[0010] US 3,867,259 (from Forgione) discloses a diagnostic test device (dry strip) for determining LDH serum concentration. The testing device contains absorbent material that accumulates the dried residue resulting from the impregnation of the absorbent material (i) with a tetrazole salt (ii) protection against the chromatographic effect, (iii) an antioxidant, (iv) diaforase and (v) nicotinamide adenine dinucleotide.

[0011] Lippenheide et al. (1995) describe LDH measurement in milk using both wet and dry chemistry. Lippenheide et al. stated that high precision, accuracy and easy operation are achieved by applying dry chemistry analysis to wet chemistry analysis, however, Lippenheide et al. does not describe the detailed structure of the device in the form of a dry strip. Despite the recommendations of Lippenheide et al. the fact is that dry strip devices available on the market today and dry strip devices described in the prior art, such as US 3,867,259 as mentioned above, consist of undifferentiated material impregnated with one or more reagents. Dry strip instruments containing undifferentiated environments combine together both storage stability and performance of the impregnated reagent (s) because optimal storage stability is almost never achieved under the same environmental conditions (e.g. pH and / or salt content) as optimal dry belt performance.

Thus, by using undifferentiated dry strip devices the material environment, i.e. washer (i) was constructed in such a way that the dry strip device exhibits acceptable (but not optimal) storage stability and acceptable (but not optimal) performance.

[0012] US 3,901,657 discloses a multilayer device suitable for colorimetric determination of the presence of a given compound in a test solution. The multilayer device comprises at least an outer layer, optionally a barrier layer, and an inner layer adjacent to the barrier layer or outer layer. The multilayer device is used for the chemical analysis used to determine morphine.

EP 1 982 182 Β1 [0013] US 4,732,736 discloses a multi-layer analytical element for the detection of hydrogen peroxide. The multilayer analytical element contains a functional material with peroxidase, a hydrogen donor and a bond in which the functional material with peroxidase and a hydrogen donor are arranged in different layers in such a way that they are separated from each other but are able to interact when in the analytical element liquid sample used. When using the analytical element, the hydrogen donor is used by the peroxidase present in the functional material with the peroxidase.

[0014] US 4,959, 305 discloses a multi-zone test device for determining an analyte in a liquid research environment. The multi-zone test contains a reagent zone, reaction zone and detection zone - the multi-zone test contains an antibody that binds to an analyte labeled with a detectable chemical group.

[0015] US 2004/219694 discloses an enzyme analysis device in a horizontal flow technique and a test kit for determining an analyte in a test sample. Furthermore, the invention relates to a horizontal flow process for the determination of analytes directly using an analyte as an enzyme substrate.

[0016] US 2005/260695 discloses compositions, kits and methods useful for detecting mastitis in animals. These factors and methods are primarily directed at the process of detecting the presence of mammary gland inflammation, including subclinical mammary gland inflammation in cows, involving the incubation of a milk sample from a cow with a factor that binds to lactoferrin, such as e.g. monoclonal antibody specific for lactoferrin, followed by detection of bound lactoferrin.

[0017] US 4,438,067 discloses a test strip for dipping and reading the colorimetric determination of solutes in a liquid. These strips consist of an inert support pad covered with a layer of polymer beads that contain reagents. The color reaction takes place in the beads themselves, which improves the sensitivity and repeatability of the tests.

[0018] US 4,454,094 discloses an indicator that aims to detect at least one test substance in a test environment, comprising a carrier and a reaction system consisting of at least a first and a second reagent

ΕΡ 1 982 182 Β1 applied separately to the carrier, said first reagent being intended to be diffused to the second reagent through a portion in the carrier soaked in the test medium during the reaction with the test substance. The indicator is characterized in that the first reagent is used in at least one limited place, while the second reagent is used in at least one place, at different distances from the place of the first reagent.

[0019] FR 2,191,734 discloses an integral analytical element that can be used in the analysis of liquids, wherein the element has at least two overlapping layers, including a distribution layer and a reagent layer, in contact with the liquid.

[0020] US 2003/073073 discloses a method and immunoanalytical device for the rapid and simultaneous detection of many microorganisms in biological fluids of dairy animals suffering from mastitis. This method is based on an horizontal immunological flow technique performed to detect antigens specific to many infectious agents that are known to cause and / or occur in cases of mastitis.

[0021] Therefore, it may be interesting to provide a dry strip device, such as a differentiated dry strip, which at the same time exhibits greater storage stability and better performance during analysis compared to dry strip devices manufactured in a conventional manner.

As a result, there is a need for an improved dry strip device, such as a differentiated dry strip device, in which storage stability and / or performance of the dry strip device would be significantly improved, thereby providing differentiated environment pads each optimized in such a way to significantly improve storage stability and performance of the reagent (s) and thus of the instrument in the form of a dry strip. This need was met by the present invention, which is discussed further below.

SUMMARY OF THIS INVENTION

ΕΡ 1 982 182 Β1 [0022] Accordingly, the object of the present invention is in a first aspect to provide an improved instrument construction in the form of a dry analyte determination strip in a sample. Preferably, the improved dry strip device relates to a differentiated dry strip device for determining an analyte in a sample.

[0023] The structure of the dry strip device consists of at least one reagent pad containing the reagent or a combination of reagents capable of reacting with the analyte, derivative of said analyte or indicator compound for said analyte to provide a detectable signal in the wetted state. At least one reagent pad provides the first environment for the reagent (s) which will allow improved stability during storage of the reagent (s) and the instrument in the form of a dry strip when not wet. In addition, the dry strip device consists of a shim that is in contact with at least one reagent shim. The shim creates a second environment for the reagent (s) when wetted, thus allowing an increased reaction rate between the analyte and reagents when wetted. In use, a sample suspected of containing analyte is applied to at least one pad with the instrument's reagents in the form of a dry strip. The sample migrates to at least one reagent pad and dissolves the reagent®, and thus, either alone or together with the reagent (s), migrates from at least one reagent pad to the regulatory pad that contacts the at least one reagent pad. The wetting pad creates a second environment in the wetted condition, allowing the reaction rate between the analyte and reagents to increase, thereby allowing the reagent and analyte, derivative of the replaced analyte or detectable compound of the replaced analyte to provide a detectable signal that can be directly or indirectly observed with a suitable instrument, apparatus or visual inspection.

[0024] In one aspect of the present invention, the device comprises:

(i) an optional solid support,

ΕΡ 1 982 182 Β1 (ii) at least one reagent pad containing the reagent or a combination of reagents capable of reacting with the analyte, derivative of said analyte or indicator compound for said analyte to provide a detectable signal when wetted, with at least one pad with the reagents provides the first environment for the reagent (s) listed, and said first environment allows better stability of the reagent (s) and the device in the form of a dry strip during storage in a non-wetted state (iii) a control pad in contact with at least one reagent pad, the control pad creating a second environment for said reagent (s) , in a wetted state, and said second environment allows the reaction rate between the analyte and reagent (s) to be increased, and in which the sample is applied to one surface of the instrument in the form of a dry strip, and a detectable signal is detected on it and in which the analyte determination is based on an enzyme-based determination and in which conditions in the first environment are achieved by adjusting the pH value to a value that deviates from the optimal pH value of the enzyme (s) and in which conditions in the second environment are achieved by adjusting the pH value to the value, which approaches the optimal pH of the enzyme (s) and in which different environments have different pH values.

[0025] In another aspect of the present invention there is provided a method of analyzing an analyte in a milk sample. The method comprises the following steps:

(i) applying a sample of milk suspected of containing analyte to the reagent pad (ii) allowing the sample to migrate to the reagent pad containing the reagent or combination of reagents capable of reacting with the analyte, derivative of said analyte or indicator compound for said analyte to provide a detectable signal in wetted condition, wherein at least one reagent pad provides the first environment for said reagent (s), and listed

EP 1 982 182 pierwsze1 the first environment allows a better stability of the reagent (s) and the instrument in the form of a dry strip during storage in a non-wet condition (iii) allowing the sample, either alone or together with the reagent, to migrate from the reagent pad to said regulating pad, wherein said regulating pad is in contact with the reagent pad and the regulating pad creates a second environment for said reagent when wetted, wherein said second environment allows the reaction rate between the analyte and reagent to be increased, and (iv) enabling the reagent and analyte, a derivative of said analyte or an indicator compound of said analyte to provide a detectable signal in which the analyte determination is based on an enzyme-based assay and in which conditions in the first environment are achieved by adjusting the pH value to the value, which deviates from the optimal pH value of the enzyme (s) and in which conditions in the second environment are provided by adjusting the pH value to a value that approaches the optimal pH value of the enzyme (s) and in which different environments have different pH values.

[0026] In yet another aspect of the present invention, there is provided a method of making a dry strip device according to the present invention. The method includes the steps of:

(i) providing a reagent pad by impregnating the first porous material with an aqueous solution containing the reagent or a combination of reagents capable of reacting with the analyte, derivative of said analyte or indicator compound for said analyte in order to provide a detectable signal in the wetted condition, wherein at least one pad with reagents provide the first environment for said reagent (s), and the said first environment allows better stability of the reagent (s) and the device in the form of a dry strip during storage (ii) then drying the reagent pad,

ΕΡ 1 982 182 Β1 (iii) providing a control pad by impregnating the second porous material with an aqueous solution creating a second environment for said wetted reagent (s), said second environment allowing the reaction rate between analyte and reagent to be increased, (iv) then drying the impregnated second porous material, and (v) contacting the reagent pad with the reagents with the adjusting pad, optionally on a solid support, to obtain a device in the form of a dry strip, in which the device in the form of a dry strip is based on an enzyme-based assay and in which conditions in the first environment are provided by adjusting the pH value to a value that deviates from the optimal pH value of the enzyme (s) and which conditions in the second environment are ensured by adjusting the pH value to the value, which approaches the optimal pH of the enzyme (s) and in which different environments have different pH values.

[0027] The present invention will be described in more detail below.

DETAILED DISCLOSURE OF THIS INVENTION [0028] The inventors of the present invention have accidentally discovered and developed a new apparatus design in the form of a dry strip that simultaneously meets the requirements of high storage stability and good performance characteristics during test or analysis. The dry strip device may have the construction of a differentiated dry strip device.

[0029] In the present context, the term "differentiated" refers to a dry strip device with different environments (e.g., different pH values) in the same dry strip. These different environments help to achieve better stability of the impregnated reagents and the dry strip during storage, and improve the reaction rate in the dry strip when analyzing the analyte. On the other hand, the term "undifferentiated" refers to a device in the form of a dry strip containing only one medium in which the impregnated reagent (s) are stored.

ΕΡ 1 982 182 Β1 [0030] The design of the new instrument in the form of a dry analyte determination strip in the sample includes: (i) optionally a solid support, (ii) at least one reagent pad containing the reagent or combination of reagents capable of reacting with the analyte, derivative of said analyte or indicator compound for said analyte to provide a detectable signal in a wetted state, with at least one reagent pad provides the first environment for the reagent (s) listed, and said first environment allows for better stability of the reagent (s) and the dry strip instrument during storage in a non-wet condition, and (iii) an adjustment pad in contact with at least one reagent pad, the adjusting pad creating a second environment for said reagent (s) ), in a wetted condition, and the latter environment allows the reaction rate between the analyte and the reagent (s) to be increased, and in which the sample is applied to one surface of the instrument in the form of a dry strip, and a detectable signal is detected on it and in which the analyte determination is based on an enzyme-based determination and in which conditions in the first environment are achieved by adjusting the pH value to a value that deviates from the optimal pH value of the enzyme (s) and in which conditions in the second environment are achieved by adjusting the pH value to the value, which approaches the optimal pH of the enzyme (s) and in which different environments have different pH values.

[0031] It may be advantageous that the method and apparatus of the present invention for determining an analyte are based on enzymatic determination. The activity of enzymes strongly depends, e.g. on changes in the pH value in the surrounding environment. Regarding the dependence on the pH value, it is generally known that each enzyme works best at a certain pH value (optimal enzyme pH) and that its activity decreases at pH values ​​above and below this point, but that at values ​​deviating from the optimal pH the enzyme activity will preserved to some extent. In addition, it is clear to one skilled in the art that this optimal pH is different for each enzyme, and that the range between these optimal pH values ​​can be very large. For example, the optimal pH value for most human enzymes is between pH 6 and pH 8,

ΕΡ 1 982 182 Β1 however, the enzyme that breaks down the protein pepsin, secreted in the stomach, is active only in an acidic environment and its optimum pH value is pH 2, while the optimal pH value for trypsin, the protein splitting enzyme secreted in the pancreas, is pH 8, 5. Therefore, during analysis, it may be important to provide a pH value as close as possible to the optimum pH value for the enzyme of analysis for fast reaction.

[0032] In the present invention, the term "first environment" refers to conditions around a reagent or reagent combination in a reagent with a wetted reagent, in which the reactivity of the reagents or combination of reagents is reduced relative to the reactivity under optimal conditions (also in the non-wetted condition). It is clear that these conditions are different depending on the enzyme present and / or the enzyme being the analyte to be determined. Optimal conditions are known to those skilled in the art and are available in the art. In a preferred embodiment of the present invention, the first environment conditions can be provided by setting the pH value to a value different from the optimal pH value for the given enzyme (s), e.g. by removing acid or base.

[0033] In a preferred embodiment of the present invention, the first environment conditions may be selected to promote stability during storage of the reagent (s) capable of reacting with the analyte to provide a detectable signal.

[0034] The storage period of the dry strip device according to the present invention can be substantially extended thanks to its new dry strip device design. The extended shelf life provided by the present invention relates to a longer shelf life without substantial loss of properties during analysis. Thus, the shelf life of the dry strip device according to the present invention may be at least 15% longer than similar conventional dry strip devices such as e.g. 20% longer, e.g. 50% longer, e.g. 75 % longer, e.g. 100% longer, e.g. 150% longer, e.g. 200% longer, e.g. 300% longer or 500% longer

ΕΡ 1 982 182 Β1 [0035] In one embodiment of the present invention, the first environment conditions are provided by setting the pH value to a value deviating from the optimum pH value for the given enzyme (s). Preferably, the deviation from the optimal value may be at least 0.5 pH units, e.g. 0.75 pH units, e.g. at least 1 pH unit, e.g. at least 1.25 pH units, e.g. at least 1, 5 pH units, e.g. at least 1.75 pH units, e.g. at least 2 pH units, e.g. at least 2.5 pH units, e.g. at least 3 pH units, e.g. at least 3.5 pH units, e.g. at least 4 pH units, e.g. at least 4 , 5 pH units, or e.g. at least 5 pH units.

[0036] In the present invention, the term "second environment" refers to the conditions in the device in the form of a dry strip around the reagents or combination of reagents provided by the adjusting pad when it is wetted, in which the reactivity of the reagents or combination of reagents (pre-impregnated with at least one reagent pad) is approaching optimal conditions for the enzyme (s) to improve the reactivity of these enzymes. That is, the shim is provided to introduce a change in the device in the form of a dry strip that transfers reagents and / or analyte from a substantially inactive state or from a state of reduced activity to a state of increased activity capable of providing reliable, repeatable and easy determination analyte.

[0037] Again, it is obvious that these optimal conditions are different depending on the enzyme present and / or the enzyme designated as analyte, and that optimal conditions are known to experts and are available in the art. In a preferred embodiment of the present invention, the conditions in the second environment can be provided by setting the pH value to a value close to the optimum pH value for the given enzyme (s), e.g. by adding acid or base.

[0038] In a preferred embodiment of the present invention, the conditions in the second environment are selected in such a way as to promote the reactivity of the reagent (s) capable of reacting with the analyte and providing a detectable signal, or in such a way as to promote the reaction rate between the analyte and

EP 1 982 182 Β1 reagent (s) capable of reacting with the analyte, providing a detectable signal.

[0039] In another embodiment of the present invention, the conditions in the second environment are provided by setting the pH value to a value that approaches the optimum pH value for the given enzyme (s). Preferably, the pH value deviates from the optimal pH value by less than 2 pH units, e.g. less than 1 pH unit, e.g. less than 0.75 pH units, e.g. e.g. less than 0.5 pH units, e.g. less than 0.25 pH units, e.g. less than 0.1 pH units, or e.g. less than 0.05 pH units.

[0040] When the analysis includes two or more enzymes with different optimal values, the second environment may deviate from the optimal conditions to ensure that all enzymes involved in the analysis promote analyte analysis. In one embodiment of the present invention, the deviation from the optimal value may be less than 2 pH units, e.g. less than 1 pH unit, e.g. less than 0.75 pH units, e.g. e.g. less than 0.5 pH units, e.g. . less than 0.25 pH units, such as less than 0.1 pH units, or e.g. less than 0.05 pH units.

[0041] In a preferred embodiment of the present invention, the provided method and instrument comprise an enzymatic analyte assay. This includes types of analysis in which one or more enzymes are impregnated in at least one reagent pad and the analyte may or may not be an enzyme. However, enzymatic analyte determination also applies to analyzes in which the analyte being determined is an enzyme, regardless of the presence or absence of an enzyme impregnated in a reagent pad. It is preferred that the analysis is of a type in which the enzyme is impregnated in at least one reagent pad and the analyte may or may not be an enzyme.

[0042] The inventors of the present invention also provided a new method of analyte analysis in a milk sample. The method comprises the following steps:

(i) applying a sample of milk suspected of containing analyte to the reagent pad (ii) allowing the sample to migrate to the reagent pad containing the reagent or combination of reagents capable of reacting with the analyte,

ΕΡ 1 982 182 Β1 a derivative of said analyte or an indicator compound for said analyte to provide a detectable wet signal, at least one reagent pad providing the first environment for said reagent (s), and said first environment allows better storage stability the reagent (s) and the instrument in the form of a dry strip (iii) enabling the sample, either alone or in conjunction with the reagent, migrating from the reagent pad to the regulating pad, wherein said regulating pad is in contact with the reagent pad, and the regulating pad creates a second environment for said reagent in a wetted condition, said second environment allowing the reaction rate between analyte and reagent to increase, and (iv) enabling the reagent and analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal in which the analyte determination is based on an enzyme-based assay and in which conditions in the first environment are achieved by adjusting the pH value to a value that deviates from the optimal pH value of the enzyme (s) and in which conditions in the second environment are ensured by adjusting the pH value to the value, which approaches the optimal pH of the enzyme (s) and in which different environments have different pH values.

[0043] The detectable signal can be any substance that can be directly or indirectly observed by any type of visual or instrumental means. Instrumental measures can be, for example, a magnetometer, spectrometer or ELISA reader. Various suitable compounds may be appropriate for use as color producing compounds. In the present invention, the color producing compound may be selected from the group consisting of chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, radioactive labels, metals, magnetic particles, dye particles, enzymes or substrates, or latex particles of organic polymers; liposomes or other vesicles containing signal generating substances and the like.

ΕΡ 1 982 182 Β1 [0044] The sample under analysis can be applied to the instrument in the form of a dry strip, either by applying the sample or part of the sample to the instrument in the form of a dry strip, or by placing the instrument in the form of a dry strip in a container containing the sample. At step (i), it is suggested to administer the sample suspected of containing the analyte, or part thereof, to the reagent pad.

[0045] Alternatively, the sample suspected of containing the analyte, or part thereof, may be administered on a separate application pad that is capable of receiving and distributing the sample (preferably substantially homogeneously) on the reagent pad and the control pad, respectively.

[0046] In the present context, the term "application pad" refers to a pad in an instrument through which a fluid sample is applied to the instrument, and which ensures rapid adsorption of the fluid sample and the rapid and consistent release of the sample into the reagent pad and / or the pad. Accordingly, the material used in the application pad may be selected from the group consisting of nitrocellulose membrane, cellulose, polymers such as nylon, polyvinyl fluoride or latexidene, glass fibers, woven fibers, non-woven fibers, and gel membranes for chromatography. Preferably, the material used in the first environment is woven or non-woven glass fiber.

[0047] When the sample is applied to the application pad, it migrates to the reagent pad and / or the control pad (steps (ii) and (iii) in the relevant lists), thereby changing the first and second environments to their wetted states. In the present context, the term "wetted" refers to contact between the reagent (s) in the reagent (s) and / or agent in the control pad and the sample whereby the reagent (s) and / or control pad become wetted or slightly wetted. The wetted effect is that dry reagents, dry chemicals and / or dry ingredients are released and dissolved (mobilized) and a reaction in the device in the form of a dry strip begins and a detectable signal is generated depending on the amount of analyte present in the sample. In one embodiment of the present invention, the application pad is part of the reagent pad and / or the adjusting pad.

Porous material

ΕΡ 1 982 182 Β1 [0048] The materials selected for use in the at least one reagent pad and / or the adjusting pad may be selected from porous materials. In the present context, the term "porous material" refers to a material that adsorbs the sample and thereby allows its migration. The selected porous material may have pores of a size that can provide a high flow rate, which causes the reagents or combinations of reagents to dissolve quickly, and which ensures good and substantially even distribution of the samples. Preferably, the porous material can be selected to provide substantial lack of retention of triglyceride rich samples. In one embodiment of the present invention, the triglyceride retention is at a level of 0%, e.g. a maximum of 1%, e.g. a maximum of 2.5%, e.g. a maximum of 5%, e.g. a maximum of 10%, as e.g. max. 15%, e.g. max. 25%, e.g. max. 50%, or most triglycerides or all triglycerides are retained, e.g. max. 75%, e.g. max. 100%.

[0049] The porous material is preferably selected from the group consisting of a nitrocellulose membrane, cellulose, a polymer such as nylon, polyvinylfluoride or vinylidene latex, glass fibers, woven fibers, non-woven fibers, gel membranes for chromatography, diatomite earth, silica gel, oxide silicon and diatomaceous earth.

[0050] In one embodiment of the present invention, the porous material in the at least one reagent pad and / or the adjusting pad may be selected from the group of materials having a pore size preferably in the range of 10 to 30,000 pm, such as in the range of 10 to 20,000 pm, e.g. in the range from 10 to 10,000 pm, e.g. in the range from 10 to 1000 pm, e.g. e.g. in the range from 10 to 500 pm, e.g. e.g. in the range from 10 to 100 pm, e.g. in range from 10 to 75 pm, e.g. in the range from 10 to 50 pm, e.g. in the range from 50 to 200 pm, e.g. e.g. in the range from 50 to 100 pm, e.g. in the range from 100 to 500 pm, e.g. e.g. in the range from 50 to 300 pm, e.g. in the range of 75 to 300 pm, such as e.g. in the range of 75 to 200 pm, e.g. in the range of 75 to 150 pm, e.g. e.g. in the range of 75 to 120 pm.

ΕΡ 1 982 182 Β1 [0051] In yet another embodiment of the present invention, the porous material in the at least one reagent pad and / or the adjusting pad may be selected from the group of materials having a suitable pore size, such as a maximum of 500 pm, for example max. 200 pm, e.g. max. 150 pm, e.g. max. 100 pm, e.g. max. 75 pm

[0052] In another embodiment of the present invention, the porous material in the at least one reagent pad and / or regulatory pad may have high protein binding capacity, e.g. in the range of 1 to

400 pg / cm², for example in the range of 1 to 250 pg / cm², such as in the range of 1 to

200 pg / cm², for example in the range of 1 to 140 pg / cm², such as in the range of 1 to

120 pg / cm², for example in the range of 1 to 100 pg / cm², e.g. in the range from 1 to pg / cm², for example in the range of 1 to 60 pg / cm², e.g. in the range from 1 to 40 pg / cm², for example in the range of 50 to 200 pg / cm², e.g. in the range from 50 to 100 pg / cm², for example in the range of 50 to 150 pg / cm², e.g. in the range from 50 to 120 pg / cm², for example in the range of 75 to 120 pg / cm², e.g. in the range from 75 to 110 pg / cm².

[0053] In another embodiment of the present invention, the porous material in the at least one reagent and / or regulatory pad may have a high protein binding capacity, e.g. a maximum of 400 pg / cm², for example a maximum of 250 pg / cm², e.g. max. 200 pg / cm², for example a maximum of 140 pg / cm², e.g. max. 120 pg / cm², for example a maximum of 100 pg / cm², e.g. max. 80 pg / cm², for example a maximum of 60 pg / cm², e.g. max. 40 pg / cm².

[0054] Accordingly, in one embodiment of the present invention, the porous material in the at least one reagent pad and / or regulating pad may be characterized by allowing sample migration at a high capillary flow rate, e.g. in the range from 50 to 500 s / 4cm, e.g. in the range from 50 to 250 s / 4cm, e.g. in the range from 50 to 200 s / 4cm, e.g. in the range from 50 to 100 s / 4cm, e.g. in the range from 50 to 75 s / 4cm, e.g. in the range from 100 to 250 s / 4cm, for example in the range from 150 to 250 s / 4cm, such as in the range from 200 to 250 s / 4cm and for example in the range from 250 to 500

ΕΡ 1 982 182 Β1 s / 4cm, e.g. in the range from 75 to 150 s / 4cm and e.g. in the range from 80 to 130 s / 4cm, e.g. e.g. in the range from 80 to 110 s / 4cm.

[0055] In another embodiment of the present invention, the porous material in the at least one reagent pad and / or the control pad may be characterized by allowing sample migration at high capillary flow rate, e.g. max. 300 s / 4cm, e.g. max. 200 s / 4cm, e.g. max. 100 s / 4cm, e.g. max. 75 s / 4cm

Preferably, the porous material used in the at least one reagent pad and / or the control pad may be the same, in at least 2 pads, e.g. in at least 3 pads, e.g. in 4 pads, e.g. at least in 5 washers.

[0057] Accordingly, for the above porous material, it may be desirable to provide an instrument for detecting the analyte in rapid analysis. In one embodiment of the present invention, the analysis time is less than 15 minutes, e.g. less than 10 minutes, e.g. less than 8 minutes, e.g. less than 7 minutes, e.g. less than 6 minutes, e.g. than 5 minutes, e.g. less than 4 minutes, e.g. less than 3 minutes, e.g. less than 2 minutes, e.g. less than 1 minute, e.g. less than 30 seconds.

Reagent Pad [0058] In the present context, the term "reagent pad" refers to one or more pads containing a reagent or combination of reagents. The reagent or combination of reagents preferably can be impregnated into the reagent pad in such a way that the reagent or combination of reagents is immobilized (a) when dry, and mobile (a) wetted.

[0059] In the present context, the term "reagent" refers to a chemical or enzyme that reacts with an analyte or derivative of said analyte or an indicator compound for said analyte, or is involved in their determination or is necessary for their determination to provide a detectable signal. A similar definition of reagent combination can be provided that relates more specifically to 2 or more reagents, such as 3 or more reagents, e.g. 4 or more reagents, such as 5 or more reagents, e.g. 6 or more reagents.

[0060] In one embodiment of the present invention, the dry strip device comprises at least 2 reagent washers, e.g. at least 3 reagent washers, e.g. at least 4 reagent washers, e.g. at least 5 reagent washers, e.g. at least 6 reagent washers. In this embodiment, reagents that react with the analyte or derivative of said analyte or indicator compound for said analyte, or are involved in their assay or are necessary for their assay to provide a detectable signal can be introduced into various reagent pads. This may improve the stability, storage properties and use of the instrument in the form of a dry band, as incompatible compounds may be contained in different reagent pads, and thus in different instrument environments in the form of a dry band.

Adjustment pad [0061] In the present context, the term "adjusting pad" refers to a pad capable of changing the sample environment containing an analyte into a second sample environment to facilitate the determination of an analyte, derivative of said analyte or indicator compound for said analyte.

[0062] In one embodiment of the present invention, the adjusting pad may contain one or more agents capable of increasing the reaction rate. Such agents ensure a change in pH by the addition of acid, base or a combination thereof.

[0063] In yet another embodiment of the present invention, the adjusting pad is in contact with the at least one reagent pad by essentially full overlap, partial overlap or by stacking adjacent the at least one reagent pad. In one embodiment of the present invention, the adjusting pad overlaps the at least one reagent pad with at least 5% reagents, such as at least 10%, e.g. at least 25%, e.g. at least 50%, e.g. at least 75%, e.g. at least 80%, e.g. at least 90%, e.g. at least 95%. In the present context, the term "substantially full overlap" refers to two separate washers (adjustment washer and at least one washer

ΕΡ 1 982 182 Β1 with reagents) stacked on top of each other. In the present context, the term "partial overlap" refers to two separate washers (regulating washer and at least one reagent washer) overlapping only partially. 100% partial overlap relates to full overlap, while 5% deviation from 100% full overlap is essentially full overlap.

[0064] In one embodiment of the present invention, the regulating pad and the at least one reagent pad are stacked adjacent. This means that the pads are arranged in contact with each other (they touch each other). An overlap of 0% (but in contact) refers to the term 'mutually adjacent', in addition, an overlap of less than 5% can be considered as being 'mutually adjacent', e.g. overlapping up to 4%, e.g. overlap in a maximum of 3%, e.g. overlap in a maximum of 2%, or e.g. overlap in a maximum of 1%.

[0065] In yet another embodiment of the present invention, the regulating pad and the at least one reagent pad may be combined in one pad, in which the reagent (s) and agent (s) capable of increasing the reaction rate can be impregnated individually and optionally can be separated with a coating. The system, method and various chemicals useful for preparing a pad containing all substances (reagent © and agent © capable of increasing the reaction rate) in one pad is described in US 4,215,995.

[0066] In one embodiment of the present invention, one or more reagents may be introduced into the adjusting pad, while second sensitive reagents, chemical compositions or analytical reagents may be transferred to a separated pad, e.g. reagent pads.

Solid support [0067] The device of the present invention may be arranged on a solid support. In the present context, the term "solid support" refers to a material that does not affect the migration of either the reaction of the liquid sample, or the reagent (s), or agents capable of increasing the reaction rate. Solid support provides stabilizing

ΕΡ 1 982 182 Β1 the basis for the analyzing instrument and provides sufficient resistance to maintain the desired shape, and does not substantially interfere with the production of a detectable signal.

[0068] In one embodiment of the present invention, the solid support material is selected from the group consisting of pipes, polymer beads, nitrocellulose strips, membranes, filters, plastic sheets and the like. [0069] Naturally, synthetic materials or modified naturally occurring materials can be used as the solid material. Such materials include polysaccharides, for example cellulosic materials such as paper and cellulose derivatives, such as cellulose acetate and nitrocellulose, silica, inorganic materials such as, for example, inactivated alumina, diatomite earth, MgSO₄, or other inorganic, finely divided materials evenly dispersed in a porous polymer matrix, wherein the matrix may contain at least one polymer such as vinyl chloride homopolymers and copolymers, e.g. polyvinyl chloride, vinyl chloride and propylene copolymer, and vinyl chloride and vinyl acetate copolymer, canvas, both natural (e.g. cotton) and synthetic (e.g. nylon), porous gels such as silica gel, agarose, dextran, and gelatin, polymer films such as polyacrylamide, and the like.

[0070] In one embodiment of the present invention, the solid support can be omitted from the device in the form of a dry strip. In this case, the dry strip device comprises at least one reagent pad and adjusting pad. When performing analyte determination in a sample using a dry strip device without a solid support, the sample can be applied to the dry strip device on one surface, and a detectable signal can be detected on the same or on the other surface of the device.

Determined analytes [0071] An instrument or method based on the above principles can be used to determine a wide range of analytes by selecting appropriate compounds

EP 1 982 182 Β1 staining known to those skilled in the art and the invention should not be limited to the examples given herein.

[0072] In one embodiment of the present invention, the analytes to be analyzed can be selected from the group consisting of protein, enzyme, fat, carbohydrate, antibiotic, steroid such as hormones, vitamins, chemical compound, hapten, cell such as bacteria or such like leukocytes, antibody, abused drugs or blood.

[0073] In one embodiment of the present invention, the analyte is an enzyme, and this enzyme is preferably selected from the group consisting of catalase, lactate dehydrogenase (LDH), alkaline phosphatase, acid phosphatase, carboxylesterase, arylesterase, β-glucuronidase, lactoperoxidase, lipase, lysozyme, xanthine oxidase, plasmin, and beta-N-acetylhexosaminidase (NAGase), prostaglandin D synthase (PGDS).

[0074] In yet another embodiment of the present invention, the analyte is a chemical compound which chemical can be selected from the group consisting of urea, triglyceride and ketone bodies such as betahydroxybutylate acetoacetate (BOHB), acetone, ascorbic acid, nitrates, urobilinogen, cholesterol and steroids such as pregnenolone, progesterone, testosterone, dihydrotestosterone, estrone, estradiol, cortisol, cortisone, aldosterone, corticosterone, androstenedione, 17a-OH-pregnenolone, 17a-OH-progesterone, 11-deoxycorticosterone, 11-deoxycortisol, and dehydroepiandrosterone, luteinizing hormone or human chorionic gonadotropin.

[0075] In another embodiment of the present invention, the analyte is a carbohydrate, which carbohydrate can be selected from the group consisting of monosaccharides such as glucose or galactose, and disaccharides such as lactose.

Samples to be analyzed [0076] In the present context, the term "sample" refers to any sample in the form of a liquid, solid or gas, and which may be liquefied during the analysis. To wet the porous material used in the shim and / or at least one reagent shim to allow migration,

ΕΡ 1 982 182 Β1 a liquid sample may be used. In addition, it is preferred that a minimum number of preparation steps for the liquid sample are required before introducing the sample into the dry strip device. In the present context, the term "preparatory stage" refers to any type of pre-treatment with a liquid sample before or after it has been introduced into the analysis device. These pre-treatments include separation, filtration, dilution, distillation, concentration, deactivation of interfering compounds, centrifugation, heating, binding, addition of reagents or chemical treatment.

[0077] In one embodiment of the present invention, the sample may be taken from a mammal, preferably the mammal is selected from the group consisting of herd animals, cows, camels, buffaloes, pigs, horses, deer, sheep, goats, domestic animals, dogs , cats and people.

[0078] In one embodiment of the present invention, the sample may be obtained from any suitable source, however it is preferred that the sample is selected from the group consisting of milk, blood, serum, plasma, saliva, urine, sweat, glass fluid contact, cerebrospinal fluid, peritoneal exudate fluid, mucus, synovium, peritoneal fluid, amniotic fluid and the like.

[0079] In addition to physiological fluids, other liquid samples can be used, such as water, food, sewage and the like samples. In addition, solid samples may be used after they have been modified into a liquid form, for example in the form of a solution, suspension or emulsion.

Coloring compound [0080] In the present context, the term "coloring compound" refers to a chemical compound capable of producing and emitting a detectable signal, such as producing a color. The intensity of this color varies depending on the concentration of the analyte present in the sample.

[0081] Examples of coloring compounds suitable for each particular analysis can be easily recognized by an expert and such coloring compounds can be

ΕΡ 1 982 182 Β1 introduced into the device in the form of a dry strip according to the present invention.

[0082] In one embodiment of the present invention, the coloring compound is selected from the group consisting of tetrazole salt; 4-aminoantypyrine / 3,5-dimethoxy-N-ethyl-N- (2-hydroxy-3-sulfopropyl) -aniline sodium; 4-aminoantypyrine / 1-naphthol-3,6-disulfonic acid sodium salt; 4-aminoantypyrine / sodium N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine; 4 aminoantypiryny/1,7-dihydroksynaftalenu; 4-aminoantipyrine / 3,5-dichloro-2hydroksybenzenosulfonianu; tetrazole violet; 3,5-dinitrobenzoic acid; copper sulfate; N-1-naphthyl-N'-diethylenediamine-oxalic acid; Fast Red TR salt; bromocresol green; bromophenol blue, Arsenazo III; 2- (3,5-dimethoxy-4-hydroxyphenol) -4,5-bis (4-dimethylaminophenyl) imidazole; pyridylase dye; magenta dye coupler; 1,5-bis (2-hydroxy-3,5-dichlorophenyl) -3-formazancarbonitrile; copper tartrate; 3-methyl-2-benzothiazolinone hydrazone; N-propyl-4- (2,6-dinitro-4-chlorobenzyl) quinoline ethanesulfonate; imidazole hydroxydiaryl; 2-methoxy-4-morpholinophenyl diazonium tetrachloride zinc salt; 3,3 ', 5,5'-tetramethylbenzidine; 4 aminofenazonu/3,5-dichloro-2-hydroksybenzenosulfonianu; prymine / hydrazone 3-methyl-2-benzothiazoline diphosphonate; 2,5-dinitrobenzoic acid; 2- (p-indophenyl) -3- (p-nitrophenyl) -5-phenyltetrazolium chloride; 3-hydroxy-1,2,3,4tetrahydrobenzo- (h) -quinoline; or any derivatives thereof.

Increasing the reaction rate [0083] As mentioned above, the regulating pad provides a second environment for said reagent (s) when wetted, allowing the reaction rate between the analyte and reagent to increase.

[0084] In one embodiment of the present invention, the increased reaction rate is provided by changing the pH value, by adding acid, base or a combination thereof.

Auxiliary Union

ΕΡ 1 982 182 Β1 [0085] Due to the complexity of the liquid samples being analyzed, in the present invention it may sometimes be beneficial to use an auxiliary compound to improve the flow and adsorption of the liquid sample in the control pad and / or the at least one reagent pad and provide rapid, repeatable and even release of reagents and agents capable of increasing the reaction rate. The excipient can be introduced into the device either by a) adding it to the reagent pad and / or the adjusting pad, alone or together with the liquid sample, b) including the auxiliary compound in at least one reagent pad and / or adjusting pad, or c ) a combination of the above.

[0086] In one embodiment of the present invention, the excipient is added to the device in the form of a dry strip before the liquid sample is added. Preferably, the excipient is a liquid.

[0087] In another preferred embodiment of the present invention, the excipient and liquid sample are added to the device in the form of a dry strip in layers. In the present context, the term "layers" refers to the division of the volume of an auxiliary compound and the volume of a liquid sample, after which the auxiliary compound and the liquid sample are added to the first medium one by one. In this case, the auxiliary compound can be added as a liquid as well as a solid compound. In one embodiment of the present invention, the excipient compound and fluid sample are independently divided into at least two volumes, providing 4 alternating layers of the excipient compound and fluid sample, e.g., the auxiliary compound and fluid sample are independently divided into 3 volumes, providing 6 alternating volumes layers of auxiliary compound and liquid sample, e.g. the excipient and fluid sample are independently divided into 4 volumes, providing 8 alternating layers of the excipient compound and fluid sample, e.g., the auxiliary compound and fluid sample are independently divided into 6 volumes, providing 12 alternating layers of the excipient compound and fluid sample, e.g. the auxiliary and fluid sample are independently divided into 8 volumes, providing 16 alternating layers of the auxiliary compound and fluid sample, e.g. excipient and liquid sample are independently divided into 10 volumes, providing 20 alternating layers of excipient and fluid

ΕΡ 1 982 182 Β1 samples, such as excipient and liquid sample, are independently divided into 20 volumes, providing 40 alternating layers of excipient compound and liquid sample.

[0088] In yet another embodiment of the present invention, the excipient may be impregnated in at least one reagent pad (s) and / or a regulating pad.

[0089] In another embodiment of the present invention, the at least one reagent pad and / or the control pad contain at least one auxiliary compound capable of improving the flow of the fluid sample.

[0090] In yet another embodiment of the present invention, the excipient provides rapid, repeatable and uniform release of the reagent (s) in the at least one reagent and / or agent pad capable of increasing the reaction rate in the control pad. In addition, the excipient provides low protein binding.

[0091] Furthermore, the excipient may provide low retention of triglyceride rich samples and / or may reduce sample viscosity.

[0092] In one embodiment of the present invention, the excipient comprises chemical components selected from the group consisting of water, surfactant, salt, metal, sugar, protein and lipid.

Preparation of the test strip [0093] The dry strip device according to the present invention may be made by any conventional method for providing dry strip devices. In a preferred embodiment, the method of preparing a dry strip device according to the present invention comprises the steps of:

(i) providing a reagent pad by impregnating the first porous material with an aqueous solution containing the reagent or a combination of reagents capable of reacting with the analyte, derivative of said analyte or indicator compound for said analyte in order to provide a detectable signal in the wetted condition, wherein at least one pad with reagents provide the first environment for said reagent (s) and said first environment

ΕΡ 1 982 182 Β1 enables better stability of the reagent (s) and the instrument in the form of a dry strip during storage (ii) then drying the reagent pad, (iii) providing a regulating pad by impregnating a second porous material with an aqueous solution containing a second medium for said reagent (s) ) in a wetted state, with said second medium allowing to increase the reaction rate between the analyte and the reagent, (iv) then drying the impregnated second porous material, (v) contacting the reagent pad with reagents with the adjusting pad, optionally on a solid support, to obtain a device in the form of a dry strip o, and in which the device in the form of a dry strip is based on an enzyme-based assay and in which conditions in the first environment are provided by adjusting the pH value to the value, which deviates from the optimal pH value of the enzyme (s) and in which conditions in the second environment are provided by adjusting the pH value to a value that approaches the optimal pH value of the enzyme (s) and in which different environments have different pH values.

[0094] At least one reagent pad and adjustment pad may be contacted by substantially full overlap of pads, by partial overlap of pads, or by placing the control pad adjacent to at least one reagent pad. In one embodiment of the present invention, the pad arrangement may be selected so as to avoid precipitation of the sample component on the upper surface of the device. Sample components that can precipitate may be selected from the group consisting of proteins, carbohydrates, fat, cells, or other component present in the sample.

[0095] In a preferred embodiment of the present invention, a first environment may be created in such a way as to promote storage of the reagent (s) capable of reacting with the analyte and providing a detectable signal - as previously described. In addition, a second environment may be created in such a way as to promote the yield of the reagent (s) capable of being generated.

ΕΡ 1 982 182 Β1 to react with the analyte and provide a detectable signal - also as previously described. Alternatively or additionally, a second environment may be created in such a way as to promote the reaction rate between the analyte and the reagent (s) capable of reacting with the analyte providing a detectable signal as previously described.

[0096] Also in an embodiment of the present invention, a first environment may be created in such a way as to favor the sample composition suspected of containing the analyte. In the event that an analyte can be found in milk, ensure that the regulating pad or at least one reagent pad contains a pH value of about 6 or less. If such a pad, having a pH value of about 6 or less, is placed on a second pad and attached to the application pad, milk proteins can coagulate and form a sediment on the upper surface of the instrument in the form of a dry strip. This sediment may cause a decrease in the detectable signal intensity. Thus, it may be beneficial if such a pad is placed below the application pad to avoid a decrease in detectable signal.

Additional Embodiments [0097] In one embodiment of the present invention, at least one reagent pad will be positioned relative to the control pad to avoid precipitation of the sample component on the upper surface of the device. Sample component (s) that may precipitate may be selected from the group consisting of proteins, carbohydrates, fat, cells, or other component present in the sample.

[0098] In cases where the analyte can be found in milk and in which activity is controlled by a change in pH, it should be ensured that the regulating pad or at least one reagent pad has a pH value of about 5 or less. If such a pad, having an approximate pH value or less, is placed on a second pad, the milk proteins may coagulate and form a sediment on the upper surface of the instrument in the form of a dry strip. This sediment may cause a decrease in the detectable signal intensity. Thus, it may be beneficial if such a pad is placed below the sample application site to avoid an undesirable decrease in detectable signal.

[0099] In an alternative embodiment of the present invention, the term "upper side" refers to the surface of the apparatus in the form of a dry strip of the present invention on which a detectable signal can be obtained either by means of a suitable instrument or apparatus or by visual inspection. This surface may be the same as the surface on which the sample is applied, or it may be a different surface.

Determination of LDH [0100] As mentioned above, the inventors of the present invention have developed a new device design in the form of a dry strip for determining analyte concentration in a sample, such an analyte may be lactate dehydrogenase (LDH) in body fluids. The dry strip device of the present invention may prove useful for the qualitative detection and quantification of LDH in a sample, where the test means include a reagent composition contained in the porous material.

[0101] The quantitative LDH assay may be important in determining mastitis that affects the structure of the mammary gland and at the same time damages the glandular epithelium and the blood-milk barrier. Hence, mastitis affects many milk components. Major components such as fat, protein and lactose are limited and a number of enzymes are changed. LDH and / or N-acetylglucosaminidase may serve as appropriate indicators of inflammation caused by mastitis. N-acetyl-β-D-glucosaminidase, also called N-acetyl glucosaminidase (NAGase), is considered one of the best markers for breast inflammation. In addition, it has been shown that other enzymes in milk, like LDH, can have a similar value and can act as an appropriate indicator of mastitis, such as NAGase.

[0102] Alternatively, the quantification of LDH can be extremely important in detecting heart disease, especially heart attacks, because after heart attacks, the concentration of LDH, e.g. in blood, clearly increases from its normal concentration. Early detection of an abnormal increase in LDH may, of course, lead to a more accurate and quick diagnosis of heart disease.

ΕΡ 1 982 182 Β1 [0103] Because early diagnosis of abnormal heart conditions is so important, the test for detecting changes in LDH blood levels must be fast and simple enough for the clinician to perform, but accurate enough to allow diagnosis without extreme error changes or false readings. Such a mechanism is represented by a new device in the form of a dry strip of the present invention. Using this new instrument in the form of a dry strip, no instrumentation is necessary and no mixing or regeneration of reagents is necessary. Therefore, the examination can be carried out in the patient's home or in the doctor's office without any special equipment.

[0104] In one embodiment of the present invention, the method of analyzing LDH in a sample may comprise the steps of:

(i) applying a sample of milk suspected of containing LDH to the reagent pad (ii) allowing the sample to migrate to the reagent pad containing the reagent or combination of reagents capable of reacting with LDH, an LDH derivative or an indicator compound for LDH to provide a detectable wet signal , wherein at least one reagent pad provides the first environment for said agent (s), and said first environment allows better stability during storage of the reagent (s) and the dry strip device in a non-wet condition (iii) allowing the sample to migrate from the reagent pad to said control pad, said control pad being in contact with the reagent pad, and the adjusting pad creates a second environment for said reagent when wetted, wherein said second environment allows an increase in the reaction rate between LDH and reagent (s) and (iv) to enable the reagent and LDH, LDH derivative or LDH indicator compound to provide a detectable signal.

[0105] In another embodiment of the present invention, the LDH assay is based on an enzyme-based assay.

ΕΡ 1 982 182 Β1 [0106] Also in an embodiment of the present invention, an increased reaction rate is provided by changing the pH. This change in pH has been described earlier.

[0107] A new device in the form of a dry strip for determining LDH concentration in a sample may include at least one reagent pad containing a porous material such as cellulose paper which contains there dried residues resulting from its impregnation in a reagent, a combination of reagents or a number of reagent materials .

[0108] The first reagent may be a tetrazole salt. This reagent is able to transfer to the instrument area in the form of a dry strip contacted with a sample a color with such a variable intensity that is representative of the LDH concentration in the serum, which is added to the indicator. These dyes are well known in the art and generally have the formula:

<img file="PL1982182T3_D0001.tif" />

Θ x

where R¹, R² and R³, individually, the same or different are aryl or substituted aryl radicals and X is an anion such as halide etc.

[0109] Examples of useful salts of this configuration include 2,3,5-triphenyl2H-tetrazole chloride; 2- (p-iodophenyl) -3- (p-nitrophenyl) -5-phenyl-2H-tetrazole chloride (INT); nitrotetrazole blue; tetrazole blue; and the like. These salts can be incorporated into the new device in the form of a dry strip at a concentration ranging from 0.05 parts to about 0.35 parts, preferably from about 0.1 parts to about 0.2 parts, based on 100 parts of the solution used, as indicated below. [0110] The second reagent, which may be included in the at least one reagent pad of the new instrument in the form of a dry strip, contains an anti-chromatographic agent that is used to prevent the chromatographic movement of the tetrazole salt above the surface

EP 1 982 182 Β1 porous material. Examples of materials that can be used for this purpose include poly (methacrylic acid), polyacrylic acid, carboxymethylcellulose, copolymers of maleic acid and methyl vinyl ether and the like. These materials are used in amounts ranging from 0.1 parts to about 3.0 parts, preferably from about 0.5 parts to about 2.5 parts, based on 100 parts of the solution used.

[0111] The third component that will be impregnated in at least one reagent pad may be an antioxidant which is used to prevent premature coloration of the tetrazole salt. Examples of suitable antioxidants include alkylated phenols such as 2,6-tertiary-butyl-p-cresol; butylated hydroxytoluene, 4-butyl catechol, octadecyl-3,5-di-t-butyl-4-hydroxyhydrocinnamate; alkylidene bisphenols such as 2,2'-methylene bis (6-t-butyl-4-methyl phenol), 4,4'-butylidene bis (6-t-butyl-3-methyl phenol); thiobisphenols such as 4,4'-thiobis (6-t-butyl-3-methylphenol), 2,2'-thiobis (6-t-butyl-4-methylphenol); polyphenols such as tetrakis [methylene (3,5-di-t-butyl-4-hydroxyhydrocinnamate)] methane, 1,3,5-trimethyl-2,4,6-tri (3,5-di-t-butyl-4-hydroxybenzyl) )benzene; esters such as ditridecyl thiodipropionate, distearyl thiodipropionate, dilauryl thiodipropionate; amines such as p-phenylenediamine with substituted diaryl or dialkyl, diphenylamine, N-phenyl-alpha-naphthylamine; organic phosphites such as dibutylofosforyn, didecylofosforyn, dioktylofosforyn, difenylodecylofosforyn, ditetradecylofosforyn, fenylodidecylofosforyn, fenyloneopentylofosforyn, tridecylofosforyn, trilaurylotritiofosforyn phosphite, triphenyl phosphite, trisnonylowy and various other well-known antioxidants such as the quinones including hydroquinone monomethyl ether, hydroquinone mono-tbutylohydrochinon. 2,5-di-t-butyl hydroquinone, toluhydroquinone, 2,5-di-tamyl hydroquinone and the like. Phenothiazine, hydroxybenzophenone, p-dimethylaminonitrosobenzene, thiodipropionic acid etc. can also be used. [0112] These antioxidants can be used in amounts ranging from 0.01 parts to 2.0 parts, preferably from about 0.02 parts to 1.0 parts in based on 100 parts of the solution and can be used in conjunction with the tetrazole salt or before or after its precipitation.

ΕΡ 1 982 182 Β1 [0113] The fourth component, which can be impregnated in at least one reagent pad, may be a diaforase which is used to catalyze the reduction of the tetrazole salt from NADH. This enzyme is well known in the art and should be used in concentrations ranging from 0.02 parts to 0.2 parts by mass and is preferably used from 0.03 parts to 0.10 parts based on 100 parts of the solution used.

[0114] The nicotinamide adenine dinucleotide, sometimes referred to hereinafter as NAD, with an admixture of a basic lactate salt such as lithium lactate, sodium lactate, potassium lactate and the like, is another ingredient that can be impregnated in at least one reagent pad. The use of NAD is well known in the art and should be used in concentrations ranging from 0.01 parts to about 0.20 parts, preferably from 0.015 parts to 0.08 parts by weight based on 100 parts of solution. Lactate salt is used in amounts ranging from 0.03 parts to about 1.5 parts, and preferably from 0.02 parts to 0.09 parts based on 100 parts of the solution used.

[0115] It is contemplated that the following reaction of the present reagents may be used in the LDH assay in the sample using the mechanism described above:

LDH

L- lactate + nad * -► Pyruvate + nadh + H *

diaphorase

NADH + NTB -► NAD * + Formazan dye [0116] The scheme of the above reaction illustrates that when a sample is added to the instrument in the form of a dry strip, the LDH contained therein can cause a reaction, which results in the reduction of the tetrazole salt and the formation of a colored indicator whose intensity is directly proportional to LDH concentration. The clinician then simply compares the resulting color with the standard color chart to determine the LDH concentration of the test serum.

[0117] In order to achieve optimal results with the new dry band device according to the present invention, it is also preferred, although not necessary, to include in the at least one reagent pad any suitable nonionic wetting agent well known in the art.

ΕΡ 1 982 182 Β1 experts to be applicable. For example, fatty alkanolamides, i.e. products of an alkanolamine reaction with fatty acids such as lauric acid or purified coconut fatty acid may be used, with suitable alkanolamines being diethanolamine, monoethanolamine, ammonisopropanolamine and the like; ethylene oxide derived materials, i.e. those derived from the reaction of ethylene oxide with alkyl phenols, where the alkyl group is octyl, nonyl or higher, long chain fatty alcohols such as tridecyl alcohol, lanolin, lecithin etc., long chain fatty acids such as tall oil, oleic acid, abietic acid etc. ., long chain fatty mercaptans, long chain fatty amines, polyoxypropylene glycol, fatty sorbitol ester; sugar esters, i.e. alcohol reaction products of the fatty acid methyl ester and sucrose or raffinose; polisorbitol; polyvinyl alcohol; methylcellulose; ethoxylated phenol / formaldehyde resins and the like. Concentrations from about 0.01 parts to about 1.0 parts of wetting agent based on 100 parts of solution are used, with wetting agents preferably being added with each component if the components are added alone or in admixture with the components if added as full system involved.

[0118] In the production of a new device in the form of a dry strip, the method used depends mainly on the reagent that is used as the antioxidant tetrazole salt. If the antioxidant is only soluble in an organic solvent, dry porous material, usually paper, may be impregnated with reagents in a series of dips. Alternatively, the reagents are impregnated in two or more different reagent washers.

[0119] In one embodiment of the present invention, an aqueous solution of the tetrazole salt may be prepared and optionally in combination with an anti-chromatographic agent and at least one reagent pad may be contacted with them and then dried e.g. in a drying tunnel or oven with forced thrust. The impregnated reagent pad or second reagent pad may then be contacted with the antioxidant solution in the organic solvent. The media is dried again. Then you can prepare a diaphorase buffer solution and

ΕΡ 1 982 182 Β1 optional carbohydrate stabilizer and one of the once impregnated, twice impregnated or three times impregnated reagent pads can be impregnated and dried in them. NAD buffer solution and basic sodium lactate and one of the treated papers or another reagent pad can be prepared, i.e. a fourth reagent pad, can be impregnated and a fourth drying completes the preparation of the test indicator [0120] In one embodiment of the present invention, the method of determining LDH in a sample can be carried out using a dry strip device having at least one reagent pad comprising:

(a) a dyeing compound, (b) a diaphase, and (c) a nicotinamide dinucleotide, and (d) a lactate salt.

[0121] Reagents (a), (b), (c) and (d) may be on a single reagent pad or on separate reagent pads, e.g. on 2 different reagent pads, e.g. 3 different hz pads reagents or e.g. on 4 different reagent pads.

[0122] In a further embodiment of the present invention, the dry strip device may further comprise at least one reagent pad containing:

(e) an agent to prevent the chromatographic effect and / or (f) an antioxidant.

[0123] Reagents (e) and (f) may be on a single reagent pad together with reagents (a), (b), (c) and (d) or on separate reagent pads, e.g. on 2 different pads with reagents.

[0124] Reagents may be separated on at least 2 reagent washers, e.g., at least 3 reagent washers, e.g., at least 4 reagent washers, e.g., at least 5 reagent washers, e.g., at least 6 washers with reagents.

ΕΡ 1 982 182 Β1 [0125] In one embodiment of the present invention, the coloring compound has been selected from the group consisting of tetrazole salt or any derivative thereof.

[0126] If wetting agents etc. are to be included, they are added during any or all impregnations to obtain uniform deposits of reagents. Suitable materials as a carbohydrate stabilizer include maltose and sorbitol as well as water soluble polymeric ethylene oxides of both high and low molecular weight, diethylene glycol and the like at concentrations ranging from 10.0 parts to about 25.0 parts, preferably about 15 , 0 parts to about 20.0 parts based on 100 parts of solution used.

[0127] In one embodiment of the present invention, a water-soluble antioxidant may be utilized, and then all reagents may be mixed together in a buffer solution at the concentrations of each ingredient as specified above, unless each is based on 100 parts of water and may be one immersion-one drying cycle used to obtain the desired test indicator.

[0128] Examples of buffers useful in any procedure include phosphate buffer, phthalate buffer, tris buffer, citrate phosphate buffer, borate succinate buffer, etc. The preferred buffer is tris buffer, i.e. 2-amino-2 (hydroxymethyl) -1, 3-propanediol in concentrations from 0.05 to 0.2M.

[0129] The color change of the test indicators prepared according to the multi-dip process may be pink to red, while the color change of the single-dip method may range from yellow to brown.

[0130] The above concentrations presented in connection with the components that can be incorporated into the new instrument in the form of a dry strip are presented with respect to solutions of these components which are saturated with at least one reagent pad and are not intended to determine the amount of each component which is finally present on at least one reagent pad. This means that impregnating the paper support with a specific concentration of a particular ingredient in the solution will not necessarily contain the same amount or percentage of ingredient present in the porous material. Nevertheless, it has been found that the above concentrations of solution may generally be sufficient to contain

ΕΡ 1 982 182 Β1 a sufficient amount of the at least one saturation reagent pad to form a functional instrument in the form of a dry strip, with the absorbent capabilities of at least one reagent pad being a feature of materials typically used for this purpose.

[0131] Although the above discussion regarding the development of this instrument in the form of a dry strip recommends saturating at least one reagent pad by immersion, sometimes, especially if a series of saturations are to be carried out, it is necessary to apply a component solution to at least one reagent pad instead of carrier dipping because prolonged dipping may tend to wash out previously supplied components.

[0132] It is obvious to a person skilled in the art that the first and second environments can be changed if another analysis is performed to determine LDH. In addition, it is also obvious to the skilled person how to optimize the first and second environments based on the knowledge derived from the concept of the present invention, i.e., providing a first environment that can be selected in such a way as to promote storage of the reagent (s) capable of reacting with the analyte and providing a detectable signal, and providing a second environment that can arise in such a way, to promote the performance of the reagent (s) capable of reacting with the analyte and providing a detectable signal, or favor the reaction rate between the analyte and reagent (s) capable of reacting with the analyte providing a detectable signal.

[0133] In a preferred embodiment of the present invention, the dry strip device is developed to measure LDH according to the reaction scheme given above for detecting LDH. In this design it may be advantageous if at least one reagent pad has a pH value of about 6.8 pH and the control pad is equipped with a pH adjusting agent capable of providing a second environment for reagents or a combination of reagents with a pH of about 8.3.

Determination of β-hydroxybutyrate (BHB)

ΕΡ 1 982 182 Β1 [0134] ΒΗΒ is formed when fat is mobilized for energy. BHB levels, with other ketone bodies, increase during starvation or malnutrition, e.g. in animals. The level is closely related to energy status if there is a high demand for glucose, i.e. during late pregnancy and lactation of herd animals, e.g. cows.

[0135] In one embodiment of the present invention, the BHB assay can be performed using the same reaction scheme as given for the LDH assay provided above.

[0136] In a preferred embodiment of the present invention, the dry strip device is developed to measure BHB according to the reaction scheme given above for detecting BHB. In this design it may be advantageous if at least one reagent pad has a pH value of about pH 6.8 and the control pad is equipped with a pH adjusting agent capable of providing a second medium for the reagent or a combination of reagents with a pH of about 8.3.

Urea determination [0137] The determination of protein utilization can be an important parameter. In cattle breeding, it is very important that animals (e.g. cows) make optimal use of the protein contained in the feed, because protein is one of the most expensive feed ingredients. Utilization depends among other things on the amount of energy and protein simultaneously present in the animal.

[0138] In one embodiment of the present invention, the urea determination can be carried out using the following reaction scheme:

urease

Urea · + · 2H₂O -► 2NH «" + CO₃²'

NH₄⁺ + Rule -&gt; NH₃NH₃ + Indicator -&gt; Dye [0139] In a preferred embodiment of the present invention, the dry strip device is developed to measure BHB according to the reaction scheme given above for detecting BHB. In this design, and using urea obtained from canavalia, it can be advantageous if at least one

ΕΡ 1 982 182 Β1 the reagent pad has a pH value of approximately pH 8.0 and the control pad is equipped with a pH adjusting agent capable of providing a second medium for the reagent or combination of reagents with a pH of approximately 6.0.

N-acetylglucosaminidase (NAGase) assay [0140] Quantitative NAGase assays can be just as important as LDH in determining mastitis, which affects the state of the mammary gland structure and at the same time damages the glandular epithelium and blood-soft barrier.

[0141] In one embodiment of the present invention, the urea determination can be carried out using the following reaction scheme:

Nagazane

4-MU-NAG

4-MU Acid + Principle —► 4-MU-phenolate dye [0142] Here, 4-MU-NAG refers to 4-methylumbelliferyl N-acetyl-beta-Dglucosaminide, 4-MU acid refers to 4-methylumbelliferone and dye 4 -MU phenolate refers to the 4-methylumbelliferone salt.

[0143] In a preferred embodiment of the present invention, the dry strip device is developed to measure NAGase according to the reaction scheme given above for detecting NAGase. In this design it may be advantageous if at least one reagent pad has a pH value of about pH 7.0 and the control pad is equipped with a pH adjusting agent capable of providing a second medium for the reagent or a combination of reagents with a pH of about 4.6.

[0144] It is obvious to the expert that the first and second environments can be changed if another analysis is performed for the determination of LDH, BHB, urea, NAGase or any other analyte. In addition, it is also obvious to the expert how to optimize the first and second environments based on the knowledge derived from the concept of the present invention, i.e. providing a first environment that can be created in such a way as to promote storage of the reagent (s) capable of reacting with the analyte and providing a detectable signal, and providing a second environment that may arise in such a way as to promote the performance of the reagent (s)

9 1 982 182 Β1 capable of reacting with the analyte and providing a detectable signal, or favoring the rate of reaction between the analyte and the reagent (s) capable of reacting with the analyte providing a detectable signal.

[0145] The idea of ​​the present invention will be further illustrated in the following non-limiting examples.

EXAMPLES [0146] Example 1. Preparation and testing of dry LDH strips.

[0147] Type 1a, single layer construction, pH 6.8

Preparation of the impregnation solution:

[0148] 1.5 g Lithium L-lactate, 3.0 g β-NAD ⁺, 150 mg polyethylene glycol 20,000 and

1.5 g of sucrose is dissolved in 120 mL of 0.1 M phosphate buffer at pH 8.0. Then 1.5 mL of a 5% solution of Tryton 100-100 is added, followed by 1.5 g of bovine serum albumin (BSA), 30 KU diaforase and 150 mg nitrotetrazole blue (NTB). A 0.1 M 8.0 phosphate buffer is added to give a total of 150 mL and the solution is mixed. The final pH of this solution is pH 6.8.

Paper impregnation [0149] The impregnation solution is transferred to a stainless steel tank. One sheet of 20x20 cm filter paper (e.g. Whatman 3MMChr) is impregnated with a pH 6.8 impregnation solution for about 10 seconds. The filter paper is then removed from the impregnation solution and allowed to flow by holding the paper vertically for about 30 seconds using a clean clamp. A total of 10 or 11 sheets can be impregnated.

drying:

[0150] The sheets of impregnated filter paper are dried in a well ventilated oven at 38-42 ° C until they dry (approx. 45 minutes). Dried filter paper can be stored in sealed plastic bags with desiccant at about 4-6 ° C until use.

Cutting:

ΕΡ 1 982 182 Β1 [0151] The impregnated sheet is cut into 5 mm wide strips e.g. in a rotary cutter, then the strips are cut into 5 mm x 5 mm squares by cutting the strips perpendicularly e.g. in a rotary cutter.

[0152] Type 1 b, single layer construction, pH 8.3

Preparation of the impregnation solution:

[0153] 1.5 g Lithium L-lactate, 3.0 g β-NAD ⁺, 150 mg polyethylene glycol 20,000 and

1.5 g of sucrose is dissolved in 120 mL of 0.1 M Tris-HCL buffer pH 9.0. Then, 1.5 mL of a 5% solution of Tryton roztworu-100 is added, followed by 1.5 g of bovine serum albumin (BSA), 30 KU diaforase and 150 mg nitrotetrazoline blue (NTB). Tris-HCL 0.1 M buffer pH 9.0 is added to give a total of 150 mL and the solution is mixed. The final pH of this solution is pH 8.3.

Paper impregnation;

[0154] The impregnation solution is transferred to a stainless steel tank. One sheet of 20x20 cm filter paper (e.g. Whatman 3MMChr) is impregnated in an impregnation solution at pH 8.3 for about 10 seconds. The filter paper is then removed from the impregnation solution and allowed to flow by holding the paper vertically for about 30 seconds using a clean clamp. A total of 10 or 11 sheets can be impregnated.

drying:

[0155] The sheets of impregnated filter paper are dried in a well ventilated oven at 38-42 ° C until they dry (approx. 45 minutes). Dried filter paper can be stored in sealed plastic bags with desiccant at about 4-6 ° C until use.

Cutting:

[0156] The impregnated sheet is cut into 5 mm wide strips e.g. in a rotary cutter, then the strips are cut into 5 mm x 5 mm squares by perpendicular cutting of the strips e.g. in a rotary cutter.

[0157] Type 2, double layer design, pH 6.8 in reagent washer, pH ~ 12 in regulating washer

ΕΡ 1 982 182 Β1 [0158] Preparation of impregnation solution for reagent pad (pH

L8);

[0159] 1.5 g Lithium L-lactate, 3.0 g β-NAD ⁺, 150 mg polyethylene glycol 20,000 and

1.5 g of sucrose was dissolved in 120 ml of 0.1 M phosphate buffer at pH 8.0. Then, 1.5 mL of a 5% solution of Tryton roztworu-100 is added, followed by 1.5 g of bovine serum albumin (BSA), 30 KU diaforase and 150 mg nitrotetrazoline blue (NTB). A 0.1 M phosphate buffer at pH 8.0 is added to give a total of 150 mL and the solution is mixed. The final pH of this solution is pH 6.8.

Impregnation of the reagent pad:

[0160] The impregnation solution is transferred to a stainless steel tank. One sheet of 20x20 cm filter paper (e.g. Whatman 3MMChr) is impregnated with a pH 6.8 impregnation solution for about 10 seconds. The reagent pad is then removed from the impregnation solution and allowed to drain by holding the paper vertically for about 30 seconds using a clean clamp. A total of 10 or 11 sheets can be impregnated.

Drying the reagent pad:

[0161] The sheets of the impregnated reagent pad are dried in a well ventilated oven at 38-42 ° C until they dry (approx. 45 minutes). Dried reagent pads can be stored in sealed plastic bags with desiccant at about 4-6 ° C until use.

Preparation of the impregnation solution for the adjusting pad (pH 12):

[0162] 50 g TRIS (2-amino-2- (hydroxymethyl) -1,3-propanediol) is dissolved in 200 mL of deionized water to provide a solution at pH ~ 12.

Impregnation of the adjusting washer:

[0163] The impregnation solution is transferred to a plastic tank. One sheet of 10x20 cm cloth (e.g. Asahi Berncot PS-2) is impregnated with an impregnation solution at pH ~ 12 for about 30 seconds. Then the shim is removed from the impregnation solution and

ΕΡ 1 982 182 Β1 placed flat on a stainless steel mesh tray. You can impregnate about 25 sheets of shims in total.

Drying the shim:

[0164] The shims are dried in a well ventilated oven at 38-42 ° C until they dry (approx. 10 minutes). Dried cloths can be stored in sealed plastic bags with desiccant at about 4-6 ° C until used.

Laminating an impregnated washer with reagents pH 6.8 and an impregnated regulating pad pH ~ 12:

[0165] A thin layer of spray glue (e.g., 3M # 75) is applied to one side of the impregnated regulating pad. A cut 10x20 cm sheet of impregnated reagent pad is placed on a flat clean surface, and a sheet of adhesive sprayed regulating pad is placed on the reagent pad, and the two layers are laminated together by applying a pressure roller. The edges of the laminated sheet are cut to get rid of the non-laminated material. Laminated sheets can be stored in sealed plastic bags with desiccant at about 4-6 ° C until use.

Cutting:

[0166] The laminated sheet is cut into 5 mm wide strips e.g. in a rotary cutter, then the strips are cut into 5 mm x 5 mm squares by perpendicular cutting of the strips e.g. in a rotary cutter. The squares are sorted so that the filter paper side is on top.

Test of performance of LDH strips types 1a, 1b and 2 [0167] The performance test was carried out as follows: A fresh LDH calibration series in milk was prepared by treating UHT milk with the enzyme 0, 100, 500 and 1000 U / L LDH (Sigma # L1378 from cow muscles) ). 10 pL milk from the calibration series was added to one strip at 25 ° C, which was then incubated at 25 ° C for 5 minutes. The development of color on the strip was assessed by using a spectrophotometer at 520 nm.

ΕΡ 1 982 182 Β1 [0168] The experimental results of measurement with 1a, 1b and 2 stripes are shown in Table 1.

Table 1. Calibration curves for type 1a, 1b and 2 strips.

<td>LDH (U / L)</td><td></td><td>Type 1a</td><td>Type 1b</td><td>Type 2</td>

<td></td><td>PH</td><td> 6.8</td><td> 8.3</td><td>"6.8 combi 12 -&gt; 8.3" (See example 3 below)</td>

<td> 0</td><td></td><td> 0.78</td><td> 0.76</td><td> 0.77</td>

<td> 100</td><td></td><td> 0.72</td><td> 0.57</td><td> 0.58</td>

<td> 500</td><td></td><td> 0.67</td><td> 0.42</td><td> 0.41</td>

<td> 1000</td><td></td><td> 0.60</td><td> 0.34</td><td> 0.33</td>

[0169] The strip type 1a, which was impregnated at pH 6.8, gives rise to a relatively shallow calibration curve, where the dynamic range is 0.18. Belt type 1b, which was impregnated at pH 8.3, gives rise to a relatively steep calibration curve, where the dynamic range is 0.42. Belt type 2, which is made up of a pad with reagents impregnated at pH 6.8 combined with a adjusting pad impregnated at pH 12, gives rise to a relatively steep calibration curve quite similar to type 1b, where the dynamic range covers 0.44.

[0170] Accelerated stability test LDH strips types 1a, 1b and 2 [0171] An accelerated stability test was carried out by storing strips of three types 1a, 1b and 2, respectively, in closed foil bags with desiccant at 37 ° C for 1 week. After this time, a performance test was carried out as described above. It is generally accepted that if the result does not change after one week at 37 ° C in closed plastic bags, the strips may have a storage time of at least 12 months at 4 ° C in closed plastic bags. Thus, the accelerated stability test gives a good indication of the actual long-term stability of the material being tested.

[0172] The experimental results obtained from the LDH strips types 1a, 1b and 2 are shown in Table 2.

Table 2. Accelerated stability of LDH belts types 1a, 1b and 2.

ΕΡ 1 982 182 Β1

<td>LDH (U / L)</td><td></td><td>Type 1a</td><td>Type 1b</td><td>Type 2</td>

<td></td><td>pH</td><td> 6.8</td><td> 8.3</td><td>"6.8 combi 12 -&gt; 8.3" (See example 3 below)</td>

<td> 0</td><td></td><td> 0.77</td><td> 0.63</td><td> 0.77</td>

<td> 100</td><td></td><td> 0.72</td><td> 0.54</td><td> 0.57</td>

<td> 500</td><td></td><td> 0.68</td><td> 0.42</td><td> 0.41</td>

<td> 1000</td><td></td><td> 0.60</td><td> 0.35</td><td> 0.33</td>

[0173] The 1 a strip type, which was impregnated at pH 6.8, gives practically the same result curve after 1 week of accelerated aging as before. The 1b strip type, which was impregnated at pH 8.3, shows a significantly shallower calibration curve after 1 week of accelerated aging compared to the same type of strips without accelerated aging. Belt type 2, which is made of a reagent pad impregnated at pH 6.8 combined with a regulating pad impregnated at pH 12, gives practically the same result curve after 1 week of accelerated aging as before.

[0174] The type of strap 2 therefore shows the same good results as the type of strap 1b and the same good storage stability as the type of strap 1a.

Example 2. Choosing the optimal pH in terms of performance and stability

a. Single-layer LDH measuring strip construction; changing the pH of the impregnation fluid.

[0175] Test 1 (phosphate buffer, final pH 6.81):

Impregnation fluid was prepared from 100 mM phosphate buffer (pH 8.0), lactate (10 mg / mL), NAD + (20 mg / mL), diaphorase (200 U / mL) and NTB (2 mg / mL) to provide final pH 6.81. Whatman 3MMChr paper was impregnated and dried in an oven at 40 ° C for 1 hour. 5x5 mm squares were cut from impregnated paper and mounted in plastic housings.

[0176] Test 2 (TRIS buffer, final pH 8.18):

Impregnation fluid was prepared from 100 mM TRIS buffer (pH 9.0), lactate (10 mg / mL), NAD + (20 mg / mL), diaphorase (200 U / mL) and NTB (2 mg / mL) to ensure final pH 8.18. Whatman 3MMChr paper

EP 1 982 182 Β1 was impregnated and dried in an oven at 40 ° C for 1 hour. 5x5 mm squares were cut from impregnated paper and mounted in plastic housings.

[0177] Standard LDH series: LDH panels were prepared with LDH Sigma (L-2525) in UHT milk with the following activities: 0 U / L, 100 U / L, 500 U / L and 1000 U / L. [0178] The strips were tested by adding 8 pL LDH standard at 25 ° C. Incubation time: 5 minutes.

Belt performance results:

[0179] The performance results are shown in Table 1.

Table 1: Performance test for tests 1 and 2.

<td rowspan="2">Test</td><td colspan="4">Reflection%</td><td>Δ0-1000</td>

<td>0 U / L</td><td>100 U / L</td><td>500 U / L</td><td>1000 U / L</td><td></td>

<td># 1, pH 6.81</td><td> 80</td><td> 76</td><td> 67</td><td> 60</td><td> 20</td>

<td># 2, pH 8.18</td><td> 65</td><td> 57</td><td> 42</td><td> 35</td><td> 30</td>

The results show that the difference in reflection coefficient in the range of 0-1000 U / L is much larger for strips prepared at pH 8.18 in TRIS buffer than strips prepared at pH 6.81 in phosphate buffer. This means that the sensitivity is higher for strips prepared at pH 8.18 in TRIS buffer than for strips prepared at pH 6.81 in phosphate buffer. Strap stability results:

[0180] The strips were kept in sealed foil bags with desiccant at 4, 30, and 37 ° C for 1 week. The strips were then tested as described above and the results are shown in Table 2.

Table 2: Test strips from the stability test from tests 1 and 2.

<td rowspan="2">Experience</td><td rowspan="2">Temp. storage ° C</td><td colspan="4">Reflection%</td>

<td>0 U / L</td><td>100 U / L</td><td>500 U / L</td><td>1000 U / L</td>

<td rowspan="3"># 1, pH 6.81</td><td> 4</td><td> 78.8</td><td> 77.3</td><td> 69.5</td><td> 63.1</td>

<td> 30</td><td> 77.7</td><td> 76.3</td><td> 69.0</td><td> 62.8</td>

<td> 37</td><td> 77.1</td><td> 73.8</td><td> 67.4</td><td> 59.9</td>

ΕΡ 1 982 182 Β1

<td rowspan="2">Experience</td><td rowspan="2">Temp. storage ° C</td><td colspan="4">Reflection%</td>

<td>0 U / L</td><td>100 U / L</td><td>500 U / L</td><td>1000 U / L</td>

<td rowspan="3"># 2, pH 8.18</td><td> 4</td><td> 64.4</td><td> 58.0</td><td> 43.1</td><td> 36.1</td>

<td> 30</td><td> 59.6</td><td> 54.8</td><td> 41.5</td><td> 34.7</td>

<td> 37</td><td> 53.1</td><td> 48.8</td><td> 38.8</td><td> 33.9</td>

Strips prepared at pH 6.81 in phosphate buffer show only slight displacement going from 4 to 30 and then to 37 ° C. In turn, strips prepared at pH 8.18 in TRIS buffer show a significant decrease in reflection value at the end of low LDH activity in the range. The results show that the stability of the strips prepared at pH 6.81 in phosphate buffer is much better than that of strips prepared at pH 8.18 in TRIS buffer.

b. Single layer LDH measuring strip construction; change in sample pH

LDH [0181] Test 3 (pH adjustment performed using TRIS buffer):

The strips were prepared according to the recommendations of Test 1 above.

Standard LDH series in milk:

Series 0 U / L: The standard UHT milk series with no LDH added but with pH corrected for discontinuous values ​​in the range 6.55 to 8.84, was prepared by mixing UHT milk with UHT milk mixed with TRIS buffer (100 mM). Discontinuous pH values ​​are: 6.55. 7.20. 7.83. 8.13. 8.33. 8.44. 8.57. 8.64. 8.72. 8.77 and 8.84.

Series 175 U / L: The standard UHT milk series with 175 U / L LDH added and the pH corrected for discontinuous values ​​in the range 6.55 to 8.84 was prepared by mixing UHT milk containing 175 mM LDH with UHT milk containing 175 mM LDH and with admixed TRIS buffer (100 mM). Discontinuous pH values ​​are given above.

350 U / L series: The standard UHT milk series with 350 U / L LDH added and the pH corrected for discontinuous values ​​in the range of 6.55 to 8.84 was prepared by mixing UHT milk containing 350 mM LDH with UHT milk containing 350 mM LDH and with admixed TRIS buffer (100 mM). Discontinuous pH values ​​are given above.

ΕΡ 1 982 182 Β1

The strips were tested by adding 10 pL of standard LDH at 25 ° C. Incubation time: 5 minutes.

Belt performance results:

[0182] The performance results are shown in Table 3.

Table 3: Test of LDH strips in milk samples corrected for various pH values ​​using TRIS buffer.

The results show that the yield improves with increasing LDH pH values ​​in milk samples. The improvement in yield usually equalizes at a pH of about 8.4

Proposal:

[0183] These tests show that the best pH conditions to ensure optimal yields for the LDH assay under current conditions are using TRIS buffer at a pH of about 8.18, in which state the storage stability proved to be poor. On the other hand, to improve storage stability, the pH should be reduced and a pH of about 6.81 has been shown to be significantly better than storage stability provided at pH 8.18.

[0184] Thus, the initial pH value of the regulating pad may be determined by a trial and error method that, combined with a pH value (pH 6.81) providing improved storage stability, will ensure optimal (or nearly optimal) performance of the strip.

[0185] In addition, the present invention has also been shown to provide a stable dry strip system or device in which minor deviations from the optimal pH value of the shim have a limited or zero effect on the dry strip device performance that results from 3F to 3K tests .

REFERENCES [0186] • US 3,867,259 (by Forgione) • Lippenheide et al. (1995). US 4,215,995 • Trevor Palmer, 2nd edition, "Understanding Enzymes" published in 1985

ΕΡ 1 982 182 Β1

US 3,901,657 US 4,732,736 US 4,959,305 US 2004/219694 US 2005/260695 US 4,438,067 US 4,454,094 FR 2,191,734. US 2003/073073

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EP 1 982 182 Β1