IE20050815U1 - A kit for colorimetric assays of food and beverage analytes - Google Patents

Abstract

ABSTRACT The invention relates to a kit for measuring the amount of analyte in a sample, comprising a tablet comprising a tetrazolium salt and an electron carrying agent; a diaphorase, and at least one enzyme active on the analyte to be measured. In some embodiments the diaphorase may be in the tablet. The electron-carrying agent may be NAD+ or NADP+, FAD or FMN. The kit according to the invention may be used for conducing colorimetric assays and in particular may find use in the wine industry. The invention also provides a tablet for use in a diagnostic kit comprising a tetrazoluim salt, and an electron carrying agent and possibly a diaphorase and other enzymes. [Fig. 1]

Description

Title A kit for colorimetric assays of food and beverage analytes Field of the Invention The present invention relates to a kit for the colorimetric assay of analytes in food and beverages. The invention also relates to a reagent composition in tablet form for colorimetric assays for use in the wine industry. In particular, this assay results in the formation ofa coloured end product, which is therefore detectable in the visible spectrum.

Background to the Invention It is well known in the food and beverage industry that samples have to be taken periodically during production to ensure optimum quality of the end products. For example in the wine industry, samples have to be taken periodically to assess the quality of the wine. In particular samples are tested at several stages during the alcoholic fennentation process to test for the presence of key analytes such as reducing sugars (D- glucose plus D-fructose) and ethanol, and during the malolactic fermentation process to test for the presence of key analytes such as L-malie acid and L-lactic acid.

Traditionally, a range of analytes of importance in defining food and beverage quality have been analysed in coupled reactions in which NADH or NADPH is formed. In these reactions, measurement of the increase in absorbance at 340 nm (ultraviolet range) due to production of NADH or NADPH gives a direct measurement of the amount of the analyte in the test sample. However, in some instances, such as reactions involved in the measurement of L—glutamate with glutamate dehydrogenase or D-sorbitol with sorbitol dehydrogenase, the equilibrium of the reaction is in favour of the analyte. In such cases. a second reaction involving diaphorase and iodonitrotetrazolium chloride (INT) has been included to utilize the NADH product and thus “pull” the reaction in the desired direction towards the products. This diaphorase/INT reaction has only been employed when it has been necessary to ensure complete reaction of the analyte.

In the measurement of L-malic acid, D-glucose, D-fructose, L-lactic acid etc. several enzymic reactions are linked with the ultimate conversion of NAD+ to NADII or NADP+ to NADPH. The amount of NADH or NADPH formed in these coupled reactions is stoichoimetric with the amount of L-malic acid, D-glucose, D-fructose, L- lactic acid etc. present in the test sample. As stated above, in some reactions, the equilibrium of coupled reactions lies in favour of the NAD+ or NADP+, so a further reaction must be included to “pull” the reaction towards formation of NADH or NADPH. One such reaction is the conversion of L-glutamic acid to 2-oxoglutarate by glutamate dehydrogenase (GIDH), shown below: (GIDH) (l) L-glutamic acid + NAD+ + H20 2-oxo lutarate + NADH + NH4+ -9 g Since the equilibrium of this deamination reaction lies markedly in the favour of the reactants, a further reaction catalysed by diaphorase is required, in which NADH reduces iodonitrotetrazolium chloride (INT) to yield an INT-formazan product (2), leading to a rapid and quantitiative conversion of L-glutamic acid to -oxoglutarate. (diaphorase) (2) INT+NADH+H+ ——————-> NAD* + INT-formazan.

The amount of INT-formazan formed in this reaction is stoichiometric with the amount of L-glutamic acid. It is the INT-fonnazan, which is measured by this increase in absorbance at 492 nm.

This diaphorase/INT reaction is well known. However it has not been widely used for several reasons. For example, in cases where an Ultraviolet (UV) spectrophotometer is readily available, there is no need to incorporate these extra reagents and analytical steps. In addition, some sample mixtures contain reducing substances, such as L- ascorbic acid in fruitjuices, or sulphur dioxide in jam, which interfere with the assay as they react with INT causing a “creep” reaction. Such samples require pre-treatment with alkaline hydrogen peroxide before assay. lE05o815 A major disadvantage of adding the diaphorase/INT reaction to assay formulations is the need for the extra steps to be carried out in the assay. In addition the light sensitivity/instability of INT in solution is disadvantageous.

In certain industrial and production situations, a need exists for an on-the-spot determination of key analytes e.g. L-malic acid and reducing sugars (D-glucose plus D- fructose). However, the particular facility may not be able tojustify expensive analytical equipment such as a UV spectrophotometer. Using an external analytical laboratory, which is the current practice, requires rapid delivery of the sample to the laboratory for assay and analysing the results. For example in the wine industry, at harvest time there may be a backlog of samples requiring analysis, so there may be a time delay in getting the results. Even having to waitjust 1-2 days is a major disadvantage to the winemaker.

This situation is not ideal. There is thus a need for a more efficient, cost-effective method for assaying key analytes, allowing the close monitoring of the process.

In certain facilities, for example, in small wineries, the production of a coloured end product, rather than one that is only detectable in the UV range, would be advantageous.

This would allow measurement of reaction products with a cheap colorimeter rather than an expensive UV spectrophotometer. Furthermore, in such situations, it is essential that the assay reagents are compact, robust and simple to employ. Moreover it is desirable that the assay format employs ready-to-use reagents in the simplest possible format and that the test kit includes components and methodology that allows the removal of substances that would interfere with the correct performance of the determination.

Object of the Invention It is thus an object of the invention to provide an assay kit, which allows accurate and . convenient detennination of the amount of analyte in a sample.

It is a further object of the invention to provide a cost-efiective and time-saving method for colorimetric assay of analytes, particularly but not exclusively, in wine and milk samples. rd U: lEo5os15 A further object is to provide an assay, which does not require expensive equipment for determination of a result.

A still further object is to provide an assay, which can be, conducted “in-house" without the need to send samples to a laboratory for determination.

It is still a further object of the invention to provide a means to assay wine and other fermentable beverages in a way that overcomes the problems associated with the prior art.

Summagy of the Invention Accordingly, the invention provides a kit for measuring the amount of analyte in a sample, comprising (a) a tablet comprising (i) a tetrazolium salt (ii) at least one electron carrying agent (b) a diaphorase, and (c) at least one enzyme active on the analyte to be measured.

The kit may further comprise a component, preferably in tablet fonn, that removes substances in the sample being analysed that interfere with the detennination (for example phenolics in red wine that interfere with measurement of L-malic acid and reducing sugars).

The kit enables the convenient and accurate detennination of the amount of analytes, affecting food or beverage quality present in a sample, to be carried out on the production floor without the need for expensive analytical equipment, such as for example, a UV spectrophotometer.

IE050815 In some embodiments the diaphorase may be incorporated into the tablet. If the diaphorase is not included in the tablet, it may be used in admixture with the specific en2yme(s) active on the analyte to be measured.

There are numerous electron-carrying agents, such as oxygenated compounds, H+, Fe3+, and other elements, metals, and compounds in a non-reduced state. Preferably, the electron carrying agents may be reversibly oxidised. Suitably, the electron~carrying agent is a universal electron carrier, such as one selected from the group consisting of NAD+, NADP+, FAD and FMN. In favourable embodiments of the invention, the electron carrying agent is a pyridine nucleotide cofactor such as NAD or NADP; the reduction potential of these cofactors are not dependant on that of a bound flavoprotein, as is the case with FAD and FMN.

Preferably, where a pyridine nucleotide cofactor is the electron-carrying agent, the kit further comprises a flavin nucleotide cofactor, such as FMN or FAD. While not being bound by theory, it is believed that the electron carrying capacity of the pyridine nucleotide cofactor is augmented and complimented by the presence of a flavin nucleotide cofactor. These embodiments and variations are equally applicable to the aspects of the invention directed towards kits, tablets and methods.

The kit may further comprise an assisting enzyme capable of removing the product produced by the enzyme active on the analyte to be measured. The assisting enzyme may be glutamate oxaloacetate transaminase in the case of L-malic acid determination.

This enzyme serves to remove oxaloacetate produced in a L-malic acid assay, which in turn serves to ‘pull’ the reaction away from the analyte.

In an alternative embodiment of the invention, the tablet of the kit further comprises ATP. ATP is required for the determination of the presence of certain analytes, for example D-glucose and D-fructose.

Suitably the tablet further comprises a flow agent, which may be water-soluble.

Preferably the flow agent comprises sodium benzoate. However, it will be appreciated by those skilled in the art that other flow agents may be used. * 30 .. lE0508t5 Preferably the tablet may comprise a bulking agent. The bulking agent may be selected from the group consisting of lactose, mannitol, maltose, sorbitol, lactitol or xylitol.

However, it will be appreciated by those skilled in the art that other flow agents may be used.

Further preferably, the tablet may comprise a disintegration agent. The disintegration agent may comprise a mixture of sodium bicarbonate and an organic acid such as citric acid, L-malic acid, D-malic acid, succinic acid, for example.

Suitably the kit further comprises a buffer solution. Suitably, the pH and composition of the buffer solution are selected to facilitate optimum activity of the included enzyme(s), as would be well known to those of skill in the art.

In a preferred embodiment, the buffer comprises surfactant selected from the group consisting of Triton X-100, polyoxyethylene ether, polyoxyethylene sorbitan or other non-ionic detergent at a concentration of approximately 0.5% (v/v). The buffer provides and maintains the correct pH for the assay. The pH will be selected depending on the active enzymes being used. [twill be appreciated by those skilled in the art that if the pH changes the enzyme(s) will be less active or unstable, either of which will impair the test method. Likewise different enzymes are most active at different optimum pHs, and accordingly the pH should be adjusted to achieve optimum activity for the enzyme used.

Suitably the analyte is selected from the group consisting of D-glucose, D—fructose, D- galactose, L-malic acid, D-malic acid, L-lactic acid, hydroxybutyric acid, D-mannitol, D-sorbitol, L-arabitol, xylitol, acetaldehyde, ethanol or L-glutamate. However, other analytes wherein coupled reactions produce NADH or NADPH may also be assayed using the kit according to the invention.

The enzyme(s) may be selected from the group consisting of hexokinase, glucose 6- phosphate dehydrogenase, phosphoglucose isomerase, L—malate dehydrogenase, glutamate oxaloacetate transaminase, D—malate dehydrogenase, L-lactate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, D-mannitol lEo5o8r5 dehydrogenase, D-sorbitol dehydrogenase, 3-hydroxybutyrate dehydrogenase, galactose dehydrogenase and glutamate dehydrogenase. It will be appreciated by those skilled in the art that other enzymes could also be used whereby the enzyme is selected according to the analyte to be assayed.

Suitably the enzymes are supplied as a suspension in ammonium sulphate or another salt solution such as lithium sulphate or dipotassium phosphate, as a solution in glycerol at approximately 50% (v/v) or as a freeze-dried (lyophilised) powder.

The tablet of the kit is used in combination with an enzyme or enzymes that in combination with NAD+ or NADP+ catalyse the production of NADH or NADPH in a linked reaction related to the concentration of the specific analyte.

Suitably the tetrazolium salt is of the type to be reduced by NADH or NADPH in the presence of diaphorase and is present in an amount sufficient to allow quantitative detection of specific analytes in a test sample.

In a preferred embodiment the tetrazolium salt comprises iodonitrotetrazolium chloride, INT. It will be appreciated by the person skilled in the art that other tetrazolium salts may also be employed.

Suitably the diaphorase is of the correct type and has an activity level to catalyse the indicator-forming reaction involving the enzyme, NADH or NADPH and a tetrazolium compound.

The invention also provides a tablet for the determination of an analyte in a food or beverage sample, the tablet comprising: (i) a tetrazolium salt (ii) at least one electron carrying agent.

The tablet may further comprise a diaphorase. Alternatively, the diaphorase does not fonn part of the tablet and instead is used in admixture with the specific enzyme(s) active on the analyte to be measured.

'EO508t5 Preferably the tablet further comprises ATP.

Suitably, the electron-carrying agent is a universal electron carrier, such as one selected from the group consisting of NAD+, NADP+, FAD and FMN. Preferably, where a pyridine nucleotide cofactor is the electron-carrying agent, the tablet further comprises a flavin nucleotide cofactor, such as FMN or FAD.

The tablet may also comprise an assisting enzyme capable of removing the product produced by the dehydrogenase. The assisting enzyme may be glutamate oxaloacetate transaminase in the case of L-malic acid determination. This enzyme serves to remove oxaloacetate produced in a L-malic acid assay, which in turn serves to ‘pull’ the reaction away from the analyde. Suitably the tablet further comprises a buffer and/or surfactant in powder form. Preferably, the buffer, when dissolved in water, or water containing surfactant, yields the correct pH and concentration of required components.

Suitably the tablet further comprises a buffer and/or surfactant in powder form.

Preferably, the buffer, when dissolved in water, or water containing surfactant, yields the correct pH and concentration of required components.

The invention also provides a method for measuring the amount of analyte in a test sample comprising the steps of: (i) adding a test tablet to buffer solution, (ii) allowing the tablet to disintegrate and dissolve, (iii) adding the test sample, (iv) taking an initial absorbance reading (A1), (v) adding an aliquot of an enzyme(s) active on the analyte and allowing the reaction to proceed, (vi), taking a final absorbance reading (A2).

Suitably, the tablet is allowed to dissolve for about 1 to 2 minutes. Preferably, once the test sample is added, the mixture is swirled to mix the components and allowed to stand lEo5oe15 for about 30 sec. Once the enzyme is added, the reaction is suitably allowed to proceed for about 5min. before the final absorbance reading is taken.

The method may also comprise the step of treating the sample with a component to remove phenolics and tannins. In the case of red wine, polyvinylpolypyrrolidone may be used for this purpose.

In alternative embodiments, allowance may be made for ‘creep reactions’ that can occur due to interfering substances in some test samples, such as red wine. When the test sample is added to the mixture of dissolved tablet in buffer, an initial absorbance reading is taken after 30 sec (A0). The solution is then stored at room temperature for 5 min and a second absorbance reading is taken (Al). [The difference (A1) - (A0) is a measure of the creep reaction]. The enzyme active on the analyte is then added and the reaction is allowed to proceed for 5 min and the absorbance taken (A2). The absorbance increase due to the analyte is [(A2) - (A.)] - (A1) - (Ao)].

Suitably the method includes spectrophotometric measurement of the INT-formazan compound at about 400-540nm.

Brief Description of the Drawings Figure 1 shows the time course of colour formation in the determination of L-malic acid using the procedure as outlined in Example I.

A. Reaction mixture containing 6.5 ug of L-malic acid.

B. Reaction mixture containing no L-malic acid.

Reaction perfonned at room temperature (approximately 22°C) under standard assay conditions as detailed in Example 1. _;Detailed¢Description of the Drawings The diaphorase/INT reaction used in the assay kit involves quantitative utilization of NADH or NADPH with stoichiometric formation of the INT-formazan complex. This reaction could potentially be applied to all assay situations where NADH or NADPH are lE05o3 formed, offering the advantages of producing a red coloured (INT-formazan) complex which can be measured with a Simple colorimeter and also, of potentially increasing the speed of the reaction by removal of NADH or NADPH.

The invention will now be described in more detail by way of the following examples.

The reagent tablet was used to assay for the presence of a variety of key analytes as outlined in the following examples.

Examples Example 1 L-Malic Acid Determination Reagent Step 1. A buffer stock solution was prepared by dissolving 13.2 g of glycylglycine (Sigma G-1002), 24 g of L-glutamate (Sigma G-1251) 18 g of sodium hydroxide and 10 mls of Triton X-100 in 1.5 litres of distilled water. The pH was adjusted to 10.0 with 4 M NaOH or 4 M HCL and 0.4g of sodium azide was added and the volume adjusted to litres.

Step 2. Tablets for the measurement of L-Malic acid were prepared by mixing 4500 Units ofdiaphorase, 176 mg oflNT, 5 g NAD+, 25 mg FAD and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Wine samples were prepared for analysis by removal of phenolics and tannins by adding 200mg of polyvinylpolypyrrolidone (PVPP) in tablet fonn to 5 ml of wine in a polypropylene tube. The tube was sealed and the suspension mixed over about 5min.

This suspension was filtered through Whatman No. 1 filter paper and the filtrate used in the assay.

Step 4. Assays were performed by adding a test tablet to 3.0 mls of buffer solution in a special test tube designed to fit directly into a colorimeter and allowing this to disintegrate over 1-2 min in subdued light with occasional agitation. An aliquot (20 uL) of test sample was added and mixed over 30 sec. An initial absorbance reading (A,) was lE0508t5 taken and recorded. An aliquot (20 uL) of L-malate dehydrogenase (15,000 U/mls) plus glutamate oxaloacetate transaminase (600) U/ml) was added and the reaction allowed to proceed at room temperature (preferably above 22°C) for approx 5 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A2) was recorded. From these absorbance values, the concentration of L-malic acid was calculated.

Figure I shows the time course of colour fomiation in the determination of L-malic acid using the procedure outlined above. In this particular example, a test tablet as described in example I was added to 3.0 mls of buffer solution as described in example 1 and allowed to dissolve completely. Aliquots (20 uL) of sample solution containing 0 to 18 ug of L-malic acid was added and the initial absorbance reading was taken. An aliquot (20 pL) of I.-malate dehydrogenase (15,000 Units/mls) in 3.2 M ammonium sulphate was then added and the increase in colour was monitored in a recording spectrophotometer. A Unit as mentioned here and throughout the patent specification, refers to an International Unit of enzyme activity.

Example 2 D-Glucose and D—Fructose Determination Reagent Step 1. A buffer stock solution was prepared by dissolving 13.2 g of glycylglycine (Sigma G—l002), 0.95 g anhydrous magnesium chloride and I0 mls of Triton X-100 in 1.5 litres of distilled water. The pH was adjusted to 10.0 with 4 M NaOH and the volume to 2 litres.

Step 2. Tablets for the measurement of D-glucose and D-fructose were prepared by mixing 230 mg of INT, 2 g NAD+, 7.4 g ATP, 20 mg FAD and flow, bulking and » disintegration agents such as sodium benzoate, lactose or mannitol, sodium bicarbonate and citric acid to‘a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 50 mg using a Manesty tablet press. lfosoeis Step 3. Wine samples were prepared for analysis by removal of phenolics and tannins by adding 200mg of polyvinylpolypyrrolidone (PVPP) in tablet form to 5 ml of wine in a polypropylene tube. The tube was sealed and the suspension mixed over about 5min.

This suspension was filtered through Whatman No. l filter paper and the filtrate used in the assay.

Step 4. Assays were performed by adding test tablet to 3.0 mls of buffer solution in a special test tube designed to fit directly into a colorimeter and allowing this to disintegrate over 1-2 min in subdued light with occasional agitation. An aliquot (20 [,LL) of test sample was added and mixed over 30 sec. An initial absorbanee reading (A.) was taken and recorded. An aliquot (20 pL) of a mixture of hexokinase (425 Units/mls), glucose 6-phosphate dehydrogenase (212 Units/mls), phosphoglucose isomerase (1000 Units/mls) and diaphorase (400 U/ml) was added and the reaction allowed to proceed at room temperature (preferably above 22°C) for approx 5 min (until there was no significant increase in absorbance over a 1 min period). This absorbanee reading (A2) was recorded. From these absorbance values, the concentration of D-glucose + D- fructose was calculated.

Alternatively, an aliquot (20 uL) of just hexokinase (425 Units/mls) and glucose 6- phosphate dehydrogenase (212 Units/mls) and diaphorase (400 U/ml) was added and the absorbance increase after approx 5 min (A2) was recorded; subsequently, an aliquot (20 pl.) of phosphoglucose isomerase (1000 Units/mls) was added and the absorbance increase was again recorded after 5 min (A3). D-Glucose was calculated from the absorbance difference A2—A. and D-fructose was calculated from the absorbance difference A3-A2.

Example 3 D-Malic Acid Determination Reagent Step 1. A buffer stock solution was prepared by dissolving 13.2 g of glycylglycine (Sigma G-1002), 3.7 g potassium chloride, 10 g of magnesium chloride.6 litres H20 and 10mL of Triton X-100 in 1.5 litres of distilled water. The pH was adjusted to 8.0 with 4 M NaOH and the volume to 2 litres.

Step 2. Tablets for the measurement of D-Malic acid were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD+, 25 mg of FAD and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding test tablet to 3.0 mls of buffer solution in a special test tube designed to fit directly into a colorimeter and allowing this to disintegrate over l-2 min in subdued light with occasional agitation. An aliquot (20 uL) of test sample was added and mixed over 30 sec. An initial absorbance reading (Al) was taken and recorded. An aliquot (20 pL) of D—malate dehydrogenase (220 Units/mls) was added and the reaction allowed to proceed at room temperature (preferably above 22°C) for approx 30 min (until there was no significant increase in absorbance over a l min period). This absorbance reading (A2) was recorded. From these absorbance values, the concentration of D-malic acid was calculated.

Example 4 L-Lactic Acid Determination Reagent Step 1. A buffer stock solution was prepared by dissolving 13.2 g of glycylglycine, 16.5 g of D-glutamate and 10 mls of Triton X-100 in 1.5 litres of distilled water. The pH was adjusted to 10.0 with 4 M NaOH and the volume to 2 litres.

Step 2. Tablets for the measurement of L-Lactic acid were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD+, 25 mg of FAD and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were perfonned by adding test tablet to 3.0 mls of buffer solution in a special test tube designed to fit directly into a colorimeter and allowing this to disintegrate over 1-2 min in subdued light with occasional agitation. An initial absorbance reading (A.) was taken and recorded. An aliquot (20 pL) of test sample was added and mixed over 30 sec. An aliquot (20 ttL) of L-lactate dehydrogenase (2000 lE0508 M lE05o315 Units/mls) was added and the reaction allowed to proceed at room temperature (preferably above 22°C) for approx 10 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A2) was recorded. From these absorbance values, the concentration of L—lactic acid was calculated.

Example 5 Ethanol Determination Reagent Step 1. A buffer stock solution was prepared by dissolving 79 g of tetrapotassium pyrophosphate (anhydrous Sigma p-8260) in 1.2 litres water and adjusting the pH to 9.0 with 8M HC1, adding 10 mls of Triton X-100 and adjusting the volume to 2 litres.

Step 2. Tablets for the measurement of ethanol were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD+ and 25mg of FAD and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding test tablet to 3.0 mls of buffer solution in a special test tube designed to fit directly into a colorimeter and allowing this to disintegrate over 1-2 min in subdued light with occasional agitation. An initial absorbance reading (Al) was taken and recorded. An aliquot (20 pL) of test sample was added and mixed over 30 sec. An aliquot (20 1.11,) of alcohol dehydrogenase (167 Units/mls) was added and the reaction allowed to proceed at room temperature (preferably above 22°C) for approx 5 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A2) was recorded. From these absorbance values, the concentration of ethanol was calculated.

Example 6 Acetaldehyde(Determination Reagent Step 1. A buffer stock solution was prepared by dissolving 79 g of tetrapotassium pyrophosphate (anhydrous Sigma p-8260) in 1.2 litres water and adjusting the pH to 9.0 with 8M HCI, adding 10 mls of Triton X-l00and adjusting the volume to 2 litres.

IE0508l5 Step 2. Tablets for the measurement of acetaldehyde were prepared by mixing 4500 Units ofdiaphorase, 176 mg ofINT, 5 g NAD+, 25mg ofFAD and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding test tablet to 3.0 mls of buffer solution in a special test tube designed to fit directly into a colorimeter and allowing this to disintegrate over 1-2 min in subdued light with occasional agitation. An initial absorbance reading (A.) was taken and recorded. An aliquot (20 nL) of test sample was added and mixed over 30 sec. An aliquot (20 p,tL) of aldehyde dehydrogenase (7.9 Units/mls) was then added and the reaction allowed to proceed at room temperature (preferably above 22°C) for approx 5 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A2) was recorded. From these absorbance values, the concentration of acetaldehyde was calculated.

Example 7 D-Mannitol Determination Reagent Step I. A buffer stock solution was prepared by dissolving 24.2 g of Trizma base, 1,5 g of bovine serum albumin and 10 mls of Triton X-100 in 1.5 litres distilled water. The pH was adjusted to 9.0 with 8 M HCl and the volume to 2 L.

Step 2. Tablets for the measurement of D—mannitol were prepared by mixing 4500 Units ofdiaphorase, 176 mg of INT, 5 g NAD+, 25mg of FAD and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding test tablet to 3.0 mls of buffer solution in a special testptube designed to fit directly into a colorimeter and allowing this to disintegrate over 1-2 min in subdued light with occasional agitation. An initial absorbance reading (A,) was taken and recorded. An aliquot (20 pl.) of test sample was added and mixed over 30 sec. An aliquot (20 i.LL) of D—mannitol dehydrogenase (3 lfosoeis Units/mls) was then added and the reaction allowed to proceed at room temperature (preferably above 22°C) for approx. 4 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A2) was recorded. From these absorbance values, the concentration of D-mannitol was calculated.

Example 8 D-Sorbitol Determination Reagent Step I. A buffer stock solution was prepared by dissolving 17.8 g of TEA, 1.8 g KCl and 10 mls of Triton X-100 in 1.5 litres of water. The pH was adjusted to 8.6 and the volume to 2 litres.

Step 2. Tablets for the measurement of D-sorbitol were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD+, 25mg of FAD and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding test tablet to 3.0 mls of buffer solution in a special test tube designed to fit directly into a colorimeter and allowing this to disintegrate over 1-2 min in subdued light with occasional agitation. An initial absorbance reading (A1) was taken and recorded. An aliquot (20 pl.) of test sample was added and mixed over 30 sec. An aliquot (50 uL) of D-sorbitol dehydrogenase (40 Units/mls) was then added and the reaction allowed to proceed at room temperature (preferably above 22°C) for approx. 15 min (until there was no significant increase in absorbance over a 2 min period). This absorbance reading (A2) was recorded. From these absorbance values, the concentration of D-sorbitol was calculated.

Example 9 Hydrogybugyric Acid Determination Reagent Step 1. A buffer stock solution was prepared by dissolving 74.2 g of TEA, 8.7 g dipotassium hydrogen phosphate and 25 mls of Triton X-100 in 4 litres of water.

The pH was adjusted to 3.6 and the volume to 5 litres. lE05o815 Step 2. Tablets for the measurement of hydroxybutyric acid were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD+, 25mg of FAD and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding test tablet to 3.5 mls of buffer solution in a special test tube designed to fit directly into a colorimeter and allowing this to disintegrate over l~2 min in subdued light with occasional agitation. An initial absorbance reading (A1) was taken and recorded. An aliquot (20 1.1L) of test sample was added and mixed over 30 sec. An aliquot (20 pL) of 3-hydroxybutyrate dehydrogenase (270 Units/mls) was then added and the reaction allowed to proceed at room temperature (preferably above 22°C) for approx. 6 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A2) was recorded. From these absorbance values, the concentration of hydroxybutyric acid was calculated.

Examgle 10 D—Galactose Determination Reagent Step 1. A buffer stock solution was prepared by dissolving 121.4 g of Trizma base (BDH cat. no. 271l95Y), plus 7.4 g of EDTA (Sigma cat. no. ED2SS) in 4.0 litres of distilled water. The pH was adjusted to 8.6 and the volume to 5 litres.

Step 2. Tablets for the measurement of D-galactose were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD+, 25mg of FAD and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding test tablet to 3.0 mls of buffer solution in a special test tubédesigned to fit directly into a colorimeter and allowing this to disintegrate over 1-2 min in subdued light with occasional agitation. An initial absorbance reading (A1) was taken and recorded. An aliquot (20 uL) of test sample was ,8 lEo5oe15 added and mixed over 30 sec. An aliquot (20 uL) of galactose dehydrogenase (100 Units/mls) plus galactose mutarotase (4.1 mg/ml) was then added and the reaction allowed to proceed at room temperature (preferably above 22°C) for approx. 5 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A2) was recorded. From these absorbance values, the concentration of galactose was calculated.

Example 11 L-Glutamic Acid Determination Reagent Step 1. A buffer stock solution was prepared by dissolving 74.2 g of TEA, 8.7 g dipotassium hydrogen phosphate and 25 mls of Triton X-100 in 4 litres of distilled water. The pH was adjusted to 8.6 and the volume to 5 litres.

Step 2. Tablets for the measurement of D-galactose were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD+ , 25mg of FAD and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding test tablet to 3.0 mls of buffer solution in a special test tube designed to fit directly into a colorimeter and allowing this to disintegrate over 1-2 min in subdued light with occasional agitation. An initial absorbance reading (A.) was taken and recorded. An aliquot (20 uL) of test sample was added and mixed over 30 sec. An aliquot (50 uL) of glutamate dehydrogenase (200 Units/mls) was then added and the reaction allowed to proceed at room temperature (preferably above 22°C) for approx. 10 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A2) was recorded. From these absorbance values, the concentration of L-glutamate was calculated.

The words “comprises/comprising” and the words “having/including” when used herein with referenceito the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. ,9 IE050815 It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub—combination.

Claims (5)

Claims

1. A kit for measuring the amount of analyte in a sample, comprising (a) a tablet comprising (i)a tetrazolium salt and (ii) at least one electron carrying agent, (b) a diaphorase, and (c) at least one enzyme active on the analyte to be measured.

2. A kit according to claim 1 further comprising additional components selected from the group consisting of FAD or FMN; an assisting enzyme capable of removing the product produced by the enzyme active on the analyte to be measured, such as glutamate oxaloacetate transaminase; ATP; polyvinlypolypyrrolidone; a buffer solution; a buffer solution comprising surfactant; an enzyme is selected from the group consisting of hexokinase, glucose 6-phosphate dehydrogenase, phosphoglucose isomerase, L- malate dehydrogenase, glutamate oxaloacetate dehydrogenase, D-malate dehydrogenase, L-lactate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, D-mannitol dehydrogenase, D—sorbitol dehydrogenase, 3- hydroxybutyrate dehydrogenase, galactose dehydrogenase and glutamate dehydrogenase.

3. A tablet for the determination of an analyte in a food or beverage sample, the tablet comprising: (i) a tetrazolium salt (ii) an electron-carrying agent and optionally comprising a diaphorase, ATP and/or FAD or FMN.

4. A kit substantially as described herein with reference to the examples and/or the accompanying drawings.

5. A tablet substantially as described herein with reference to the examples and/or the accompanying drawings. ' 5