Stabilization of NADPH or NADH in ammonia detection assays

Abstract

The present disclosure relates to the biochemistry of reagents for detecting ammonia in liquid samples. Specifically, the present disclosure relates to technical improvements to enzyme-based ammonia tests that can be used to analyze plasma samples collected from patients in a clinical setting, among other uses. In this regard, the stability of reagents containing NAD(P)H is improved, thereby extending the shelf life and enabling the detection of ammonia. In an exemplary reagent, an enzymatic reaction is used to remove ammonia released due to the decay of NAD(P)H, and the enzymatic reaction converts ammonia using GLDH, NAD(P)H, and 2-oxoglutarate, thereby forming L-glutamate, NAD(P)H, and 2-oxoglutarate in the reagent containing NAD(P)H. + and H2O.

Description

Technical Field

[0001] The present disclosure relates to the biochemistry of reagents for detecting ammonia in liquid samples. In particular, the present disclosure relates to a technical improvement in an enzyme-based ammonia test that can be used to analyze plasma samples collected from patients in a clinical setting, among other uses. In this regard, the stability of reagents containing NAD(P)H is improved, thereby extending the shelf life and enabling the detection of ammonia. In an exemplary reagent, an enzymatic reaction is used to remove ammonia released due to the decay of NAD(P)H, and the enzymatic reaction converts ammonia using GLDH (glutamate dehydrogenase), NAD(P)H, and 2-oxoglutarate, thereby forming L-glutamate, NAD(P)H, and 2-oxoglutarate in the reagent containing NAD(P)H. + and H2O. Background Art

[0002] In higher animals, especially humans, ammonia is produced primarily in the gastrointestinal tract by the metabolism of nitrogenous compounds. Excess ammonia can be toxic to the central nervous system. The Krebs-Henseleit urea cycle provides a method for disposing of ammonia by metabolizing it to urea in the liver. Hyperammonemia in human infants can result from genetic defects in urea cycle enzymes or be acquired through acute (as in Reye's syndrome) or chronic (as in cirrhosis) liver disease. In adults, elevated ammonia levels aid in the diagnosis of liver failure or hepatic encephalopathy caused by advanced liver diseases such as viral hepatitis or cirrhosis. Ammonia is therefore an important clinical parameter and its in vitro determination is technically necessary.

[0003] In 1963, Kirsten et al. introduced an enzymatic method for ammonia determination based on the action of glutamate dehydrogenase (Kirsten E et al. Biochem Z 337 (1963) 312-319). Although the enzymatic method proved to be highly specific and utilized a direct assessment based on the molar absorbance of NADH, several problems were encountered, including difficulties in stabilizing the final reaction.

[0004] The improvement of this technical method is the improvement of the Kirsten reaction by Da Fonseca-Wollheim. The original enzymatic method is to add ADP to the reaction mixture, use NADPH instead of NADH to eliminate the interference of the reaction of endogenous LDH with endogenous pyruvate, and replace the deproteinized supernatant with plasma (Da Fonseca-Wollheim FZ Klin ChemKlin Biochem 11 (1973) 421-425).

[0005] JP03614967B2 discloses using a combination comprising 2-oxoglutarate, GLDH, 6-phosphate glucose, 6-phosphate glucose dehydrogenase and NADPH to achieve the removal of ammonium from a sample.

[0006] Chenault KH & Whitesides GM Appl Biochem Biotechnol. 14 (1987) 147-197 reported different methods for the regeneration of the nicotinamide cofactor.

[0007] An exemplary embodiment of the Da Fonseca-Wollheim improvement in the field of clinical chemistry and in vitro diagnostics is the NH3 (ammonia) determination kit (including MODULAR P, cobas c platform; for example Roche Cat. No. 11877984216, with Cat. No. 20751995190, 20752401190, 20753009190 as calibrator) for quantitative determination of ammonia in plasma. The kit used includes reagent containers containing first and second aqueous working solutions (also referred to as R1 and R2).

[0008] The assay principle utilizes a reaction catalyzed by glutamate dehydrogenase (GLDH) (EC 1.4.1.3). In aqueous solution, GLDH catalyzes the reaction of 2-oxoglutarate with NH4 + and reductive amination of the co-substrate NADPH to form glutamate and NADP + In the diagnostic assay workflow, a plasma sample is mixed with aliquots of R1 and R2 and incubated. After incubation, the co-substrate NADP is oxidized + The amount is determined photometrically by measuring the difference in absorbance of NADPH before and after addition to the sample, and the corresponding amount of ammonia in the sample is determined using calibration data collected in a separate measurement.

[0009] In order to ensure the stability and shelf life of the reactive compounds involved, their aqueous solutions need to be stored under appropriate conditions. Specifically, GLDH becomes increasingly unstable as the pH increases. If GLDH is stored under alkaline conditions above pH 10, it is technically impractical for diagnostic assays. However, a pH below pH 9 allows GLDH to have sufficient stability, shelf life, and usability. Therefore, in the exemplary In the NH3 (ammonia) assay kit, the working solution R1 is an aqueous solution buffered at pH 8.3 (ie, below pH 9), which is provided by the manufacturer as a ready-to-use liquid reagent in the commercially available assay kit.

[0010] In contrast, NADPH (and similarly NADH) is sufficiently stable in solutions at pH values ​​above pH 10. However, NAD(P)H exhibits decay over time in aqueous solutions, even at pH values ​​above pH 10. To counteract this instability, The NH3 (ammonia) assay kit comes with the dry components of R2, including NADPH. NADPH remains stable when stored as a dry substance. Prior to use, the R2 working solution is prepared by dissolving the dry NADPH in an aqueous buffer, thereby forming an aqueous solution (R2) that is ready for subsequent (preferably immediate) consumption in a diagnostic assay workflow to determine NH3 in a sample. Since this requires additional labor, a stable, ready-to-use reagent with NADPH is desired.

[0011] Due to the decay of NADPH, ammonia is formed as a product. For this particular reason, there is a technical need to counteract the decay of NADPH in the reagents in the assay for the determination and quantification of ammonia in the sample. As a result, over time, NH3 accumulates in the R2 working solution due to the decay of NADPH, with the risk of causing falsely elevated measured values.

[0012] In view of the above technical needs, the compositions, kit methods and uses described in the present disclosure are designed to provide improvements. Based on the teachings detailed herein, the assay for determining ammonia in a liquid sample can be advantageously improved. Surprisingly, the shelf life of aqueous reagents containing NAD(P)H can be extended even under harsh conditions. Summary of the invention

[0013] As a first aspect related to all other aspects and embodiments of the present disclosure, reported herein is an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample, the kit comprising 2-oxoglutarate, glutamate dehydrogenase (=GLDH) capable of reacting with NAD(P)H as a co-substrate, and NAD(P)H, wherein the kit comprises a first container and a second container, wherein the first container comprises a first aqueous reagent having a first amount of GLDH, the first reagent having a pH capable of maintaining the enzymatic activity of GLDH, wherein the second container contains a second aqueous reagent having NAD(P)H, 2-oxoglutarate, and a second amount of GLDH, wherein the GLDH enzymatic activity per mL of the first reagent is higher than the GLDH enzymatic activity per mL of the second reagent, and wherein the pH of the second reagent is capable of maintaining the enzymatic activity of GLDH in the second reagent to convert ammonia, NAD(P)H and 2-oxoglutarate, thereby enabling the formation of L-glutamate, NAD(P)H and 2-oxoglutarate in the second reagent. + and H2O.

[0014] As a second aspect related to all other aspects and embodiments of the present disclosure, a method is reported herein for providing an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample, the kit comprising two different aqueous reagents, the reagents comprising 2-oxoglutarate, glutamate dehydrogenase (=GLDH) capable of reacting NAD(P)H as a co-substrate, and NAD(P)H in an aqueous solution, the method comprising the steps of preparing a first reagent having a pH capable of maintaining the enzymatic activity of GLDH by dissolving GLDH in an aqueous solution, preparing a second reagent by dissolving 2-oxoglutarate, GLDH and NAD(P)H in an aqueous solution, and adjusting the pH of the second reagent to allow the enzymatic activity of GLDH in the second reagent to be maintained to convert ammonia, NAD(P)H and 2-oxoglutarate, thereby allowing L-glutamate, NAD(P)H and glutamate to be formed in the second reagent. + and H2O, providing the first reagent and the second reagent in separate containers, and combining the containers in a kit of parts, thereby providing an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

[0015] As a third aspect related to all other aspects and embodiments of the present disclosure, herein is reported the use of the assay kit according to the first aspect or the assay kit obtained by implementing the method of the second aspect for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

[0016] As a fourth aspect related to all other aspects and embodiments of the present disclosure, this document reports an assay kit according to the first aspect or an assay kit obtained by implementing the method of the second aspect, wherein the concentration of ammonia in the second reagent is about 1.5 μM or lower, more specifically from about 0.01 μM to about 1.5 μM.

[0017] As a fifth aspect related to all other aspects and embodiments of the present disclosure, a mixture is reported herein, which comprises (i) an aqueous liquid sample that appears to contain ammonia, and (ii) a second reagent of an assay kit, including an assay kit according to the first aspect or an assay kit obtained by implementing the method of the second aspect.

[0018] As a sixth aspect related to all other aspects and embodiments of the present disclosure, an automated device capable of forming a mixture according to the fifth aspect is reported herein, wherein the device is combined with (i) an aqueous liquid sample in a sample container, and (ii) an assay kit according to the first aspect or an assay kit obtained by implementing the method of the second aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 NADP + and the chemical formula of NADPH

[0020] Figure 2 NADP as reported in De Ruyck J. et al. Chem Phys Lett 450 (2007) 119-122 + and experimental UV / vis spectra of NADPH.

[0021] FIG. 3A to FIG. 3C According to the teachings of this report, a working description of an improved diagnostic assay for ammonia detection and quantification is described.

[0022] 4A to 4H Molecular structure description of the oxidized form of GLDH's co-substrate: (A) NAD + , (B) NADP + , (C) 3-acetylpyridinium-NAD + , (D) 3-acetylpyridine-NADP + , (E) 3-formylpyridine-NAD + , (F) 3-formylpyridine-NADP + , (G) nicotinamide-NAD + , (H) thionicotinamide-NADP + .

[0023] Figure 5 Graphical representation of the experimental results listed in Table 1 (see Example 4 and Table 1).

[0024] Figure 6 Comparison of (i) the original second reagent (R3) [solid line] working solution (containing NADPH) as described in Example 2 and (ii) the improved working solution R3' (an improved second reagent according to the present disclosure) wherein NADPH is replaced by NADH [dashed line], see Example 5 for more details. Extinction readings of NADPH [solid line] and NADH [dashed line]. DETAILED DESCRIPTION

[0025] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0026] In this detailed description, references to "one embodiment," "an embodiment," or "in an embodiment" mean that the referenced features are included in at least one embodiment of the technology in accordance with all aspects of the present disclosure. Furthermore, separate references to "one embodiment," "an embodiment," or "an embodiment" do not necessarily refer to the same embodiment; however, these embodiments are not mutually exclusive unless otherwise stated and unless otherwise apparent to one skilled in the art. Thus, the technology in accordance with the present disclosure in all its aspects may include any kind of combination and / or integration of the embodiments described herein.

[0027] Unless the context clearly indicates otherwise, the terms "a", "an", and "the" generally include plural references. As used herein, "plurality" is understood to mean more than one. For example, a plurality refers to at least two, three, four, five or more. Unless specifically stated or obvious from the context, as used herein, the term "or" is understood to be inclusive.

[0028] It will be further understood that when the root terms "include" and / or "have" are used in this specification, they specify the presence of the features, matters, integers, steps, operations, elements and / or components stated, but do not exclude the presence or addition of at least one other feature, integer, step, operation, element, component and / or group thereof. In a similar manner, "with" also specifies the presence of the stated features, etc.

[0029] As used herein, the terms "comprises / comprising / contains / containing / includes / including", "has / having" or any other variation thereof are intended to cover a non-exclusive inclusion, i.e., to represent an open list of features. For example, a process, method, article, or apparatus that includes a list of features is not necessarily limited to only those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus. In contrast, "consists of / consisting of" or any other variation thereof specifies a closed list of features. Notably, a closed list of a given feature is understood to represent a specific embodiment of the open list of those features.

[0030] As used herein, unless expressly stated otherwise, "or" refers to an inclusive or rather than an exclusive or. For example, any of the following may satisfy condition A or B: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0031] As used herein, "substantially," "relatively," "generally," "typically," and "approximately" are relative modifiers intended to indicate permissible variations from the properties so modified. They are not intended to limit the absolute values ​​or properties to which they are modified, but rather to approximate or approximate such physical or functional properties. Unless specifically stated or apparent from the context, as used herein, the term "about" is understood to be within the normal tolerance range of the art, such as within 2 standard deviations of the mean. "About" may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the value being described. Unless the context clearly states otherwise, all numerical values ​​provided herein may be modified by the term "about."

[0032] "Ammonia" is a well-known compound of nitrogen and hydrogen with the molecular formula NH3. It is soluble in water, and its aqueous solution is alkaline. Ammonia is a base with a constant pK b A base of 4.76. Thus, in aqueous solution, a certain amount of ammonia molecules react with water molecules to produce ammonium ions (="ammonium") and hydroxide ions. It should be understood that, unless expressly stated otherwise, to the extent that ammonia is present in aqueous solution, it is in a state of reaction equilibrium with ammonium.

[0033] 2-Oxoglutaric acid (=α-ketoglutaric acid) is one of the two keto derivatives of glutaric acid. Its anion 2-oxoglutarate (=α-ketoglutarate) is an important biological compound. It is a keto acid produced by the deamination of glutamate and is an intermediate of the Krebs cycle.

[0034] Glutamate dehydrogenase (=GLDH) is an enzyme present in the mitochondria of most microorganisms and eukaryotes. GLDH activity is able to convert L-glutamate into α-ketoglutarate and vice versa. It should be understood that all aspects and embodiments of this report relate to GLDH (EC 1.4.1.3) that is able to react with NAD(P)H as a co-substrate.

[0035] Nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ) are co-substrates of GLDH. They consist of an AMP (adenosine monophosphate) molecule covalently linked to nicotinamide mononucleotide, i.e. a dinucleotide. NADP + With NAD +AMP differs from NAD in that an additional phosphate group is present on carbon two of the ribose ring. These molecules are diffusible cosubstrates that participate in oxidation / reduction (= redox) reactions. + and NADP + is the oxidized form of the corresponding cosubstrate. When reduced, the carbon four of the nicotinamide ring accepts a hydride (H - ) ion, one proton and two electrons. NAD+ / NADP+ always undergoes two electron oxidation or reduction. NADH and NADPH are abbreviations for the reduced forms. Regarding GLDH enzyme activity, not only NADH and NADPH can serve as substrates, but also their functional analogs. 4A to 4H For the purpose of this report, it is hereby stated that the teachings described in all aspects and embodiments can also be advantageously implemented with any of the functional analogs, provided that the specific GLDH used is capable of reacting the reduced form of the corresponding functional analog with 2-oxoglutarate in a redox reaction to produce the oxidized form of the functional analog.

[0036] For the purposes of this disclosure, an "assay kit" is understood to be a composition of components combined for the purpose of performing an assay. Typically, the assay involves the detection and determination (quantification) of an analyte in a sample, such as, but not limited to, the analyte ammonia in plasma as the material of the sample. Importantly, the meaning of an "assay kit" refers to a composition of components, one or more components included in the kit being used up before use. In a specific embodiment of a container encapsulating (containing) a liquid reagent, it should be understood that the container is closed and optionally sealed before use. The container is opened before use, wherein the seal (if present) is broken. The aspects and embodiments reported in this disclosure relate to an assay kit as a kit of components comprising at least two components (A component and B component). Other components may also be present in an assay kit according to the present disclosure. Each component typically includes one or more components. When providing a kit of components, if possible, it is avoided to combine the components in the components, where chemical reactions may occur between the components to form undesirable products, especially during the storage of the kit of components. Therefore, the kit of components is typically provided in a manner in which these reactive components are separated from each other at least during storage to avoid undesirable reactions. While the technical issues arising from the undesired reaction of NAD(P)H are specifically addressed, it is understood that all other components of the assay kit components as disclosed herein are in an appropriate environment, such as (without limitation) the reagents are enclosed in a housing (container) that prevents evaporation of the liquid, the enzyme and substrate are provided in a solvent, which is configured to allow use according to the purpose of the assay kit and other measures accepted in the art. The assay kit also typically includes packaging materials and documents with information about the intended use of the assay kit and the storage conditions recommended or required before using the kit. With respect to the packaging materials, specific embodiments thereof allow the user to identify a kit that has been opened and therefore may not be in its original state after manufacture, for example, by the original seal.

[0037] The findings reported in the present disclosure relate, on the one hand, to the improvement of the Kirsten reaction by Da Fonseca-Wollheim. The original enzymatic method eliminates the interference caused by the reaction of endogenous LDH with endogenous pyruvate by adding ADP to the reaction mixture, using NADPH instead of NADH, and replacing the deproteinized supernatant with plasma (Da Fonseca-Wollheim F. ZKlin Chem Klin Biochem 11 (1973) 421-425). However, on the other hand, the findings of the present disclosure relate more generally to the stabilization of aqueous solutions of NADPH and NADH, in particular for the purpose of determining ammonia, wherein the relevant detection assay is based on the enzymatic activity of glutamate dehydrogenase.

[0038] Typically, ammonia-specific detection reagents are selected and designed to detect both

[0039] i) quantitatively reacting the ammonium present in the sample (and thus the ammonia in a reaction equilibrium with the ammonium in the aqueous solution), and

[0040] ii) producing a detectable reaction product in a stoichiometric amount reflecting the amount of ammonia reacted.

[0041] A well-established assay principle utilizes the following reaction [Reaction 1], which is catalyzed by glutamate dehydrogenase (GLDH). In aqueous solution, GLDH catalyzes the reaction of 2-oxoglutarate with NH4 + and reductive amination of NADPH to form glutamate and NADP + .

[0042] [Reaction 1] GLDH

[0043] NH4 + +2-oxoglutarate+NADPH→L-glutamate+NADP + +H2O

[0044] The co-substrate in [Reaction 1] is NADPH, which is the reduced form of nicotinamide adenine dinucleotide phosphate (CAS No. 53-59-8); the corresponding oxidized form is NADP + Alternatively, the cosubstrate may be NADH, which is the reduced form of nicotinamide adenine dinucleotide (CAS No. 58-68-4); the corresponding oxidized form is NAD + The alternative enzymatic reaction is similar to the following [Reaction 2].

[0045] [Reaction 2] GLDH

[0046] NH4 + +2-oxoglutarate+NADH→L-glutamate+NAD + +H2O

[0047] As described above, unless otherwise expressly stated, to the extent that ammonia is present in aqueous solution, it is in a state of reaction equilibrium with ammonium. For the purposes of this disclosure, any one of [Reaction 1] and [Reaction 2] (alone or in combination) is also referred to as a "detection reaction". In addition, any one of NADPH and NADH c is also referred to as a "reduced co-substrate". In addition, NADP + and NAD + Any of the above is also referred to as an "oxidized co-substrate".

[0048] Importantly, both detection reactions produce the corresponding oxidized cosubstrate in stoichiometric amounts, reflecting the amount of ammonium reacted. The cosubstrate strongly absorbs UV light, and the difference between the reduced and oxidized cosubstrates can be detected photometrically. Therefore, the difference in absorption spectra between the oxidized and reduced forms of the cosubstrate makes it simple to measure this conversion in an enzyme assay of [Reaction 1] or [Reaction 2].

[0049] Therefore, NADH and NADPH are used with great advantage as co-substrates in a variety of quantitative assays based on enzyme-catalyzed redox reactions, in particular assays for assessing the concentration of ammonia in liquid aqueous samples. There is a stoichiometric relationship that for each enzyme-catalyzed redox reaction involving one ammonium ion, one NAD(P)H molecule is converted to NAD(P) + . Spectrophotometric measurements allow quantitative detection of any changes in the initial amount of NADH or NADPH initially present in the reaction. With respect to [Reaction 1] and [Reaction 2], by detectably forming the corresponding oxidized form as a reaction product in stoichiometric amounts, the spectrophotometric measurement of the corresponding oxidized form reflects the amount of ammonium (and ammonia) reacted. The co-substrate and acceptor compound 2-oxoglutarate are present in molar excess, and the chemical equilibrium is shifted towards the product side (L-glutamate + NAD(P) + +H2O). As a result, all available ammonia is eventually converted, i.e., reacts quantitatively, thereby producing equimolar amounts of NAD + or NADP + .

[0050] NAD(P)H molecules in aqueous solution are unstable, i.e., they show a tendency to decay, thereby releasing ammonia. Example 3 lists Experiments #1, 3, 4 and 5 in Table 1, which illustrate that this is indeed the case for aqueous solutions of NAD(P)H with ammonia release over time. The technical problems caused by this ammonia release decay of the co-substrate are particularly evident in cases where aqueous solutions of NAD(P)H are to be used as reagents to detect ammonia in samples. The lower the concentration of ammonia in the sample, the greater the risk posed by aqueous reagents containing NAD(P)H, as quantitative measurements of ammonia may be falsely elevated.

[0051] However, the inventors were able to demonstrate, and also document in this disclosure, that even under suboptimal conditions (specifically at a pH above 9 or even higher), a certain proportion of GLDH remains active in the presence of 2-oxoglutarate and NAD(P)H. The impact of this discovery can be demonstrated in a non-limiting manner by Roche Diagnostics NH3 diagnostic assay is used as an example. The NH3 assay uses two different reagents, R1 and R2, which are mixed with a sample suspected of containing ammonia. R1 contains 2-oxoglutaric acid as a ready-made liquid reagent. R2 is freshly prepared from solid materials and aqueous solution just before use and contains NADPH, GLDH and 2-oxoglutaric acid after preparation. Rather than including two ready-to-use assay reagents, the NH3 diagnostic assay kit includes only one. The kit includes additional vials with liquid contents and solid materials that require manual preparation of the R2 reagent prior to use.

[0052] Therefore, a first aspect related to all other aspects and embodiments of the present disclosure is a test kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample, the test kit comprising 2-oxoglutarate, glutamate dehydrogenase (=GLDH) capable of reacting with NAD(P)H as a co-substrate, and NAD(P)H, wherein the test kit comprises a first container and a second container, wherein the first container comprises a first aqueous reagent having a first amount of GLDH, the first reagent having a pH capable of maintaining the enzymatic activity of GLDH, wherein the second container comprises a second aqueous reagent having NAD(P)H, 2-oxoglutarate, and a second amount of GLDH, wherein the GLDH enzymatic activity per mL of the first reagent is higher than the GLDH enzymatic activity per mL of the second reagent, and wherein the pH of the second reagent is capable of maintaining the enzymatic activity of GLDH in the second reagent to convert ammonia, NAD(P)H, and 2-oxoglutarate, thereby enabling the formation of L-glutamate, NAD(P)H, and 2-oxoglutarate in the second reagent. + and H2O.

[0053] It is important to understand that both the first reagent and the second reagent in the assay kit provided herein do not require a separate manual preparation step prior to use. Thus, in one embodiment of the assay kit, the kit may comprise only (more specifically comprise) two containers containing two aqueous solutions, wherein either of the two solutions comprises one or more compounds selected from NAD(P)H, GLDH and 2-oxoglutaric acid. In one embodiment, NAD(P)H is present only in the second reagent.

[0054] In one embodiment, the pH of the first agent is from pH 7 to pH 9, and more specifically between pH 8 and pH 9. A specific embodiment is the pH value of the first agent, wherein the specific value is selected from 8.5 to 8.9.

[0055] In particular, with regard to the second reagent, a central finding underlying this report is that, in order to remove ammonia from a reagent containing NAD(P)H, GLDH can be used under conditions that are considered technically unsuitable. Thus, conditions can be selected for the NAD(P)H reagent that are most favorable to the stability of NAD(P)H, i.e., to reduce the tendency to release ammonia. In addition, the presence of the GLDH enzyme renders the reagent ammonia-free. An important factor in this context is the pH of the reagent, which is selected to be substantially alkaline. Thus, in one embodiment, the pH of the second reagent is from pH 8 to pH 11, and more particularly between pH 8 and pH 10, and even more particularly between pH 9 and pH 10.

[0056] Example 2 illustrates the newly developed NH3L2 R1 reagent provides most of the GLDH enzyme activity for ammonia detection in the sample. It is noteworthy that R1 reagent does not contain 2-oxoglutaric acid. This is an example of an embodiment in which 2-oxoglutaric acid is only present in the second reagent.

[0057] The newly developed second (R3) reagent contains a smaller portion of GLDH, but is sufficient to maintain the ammonia scavenging mechanism. Therefore, in one embodiment, the ratio between the GLDH enzyme activity present in each mL of the first reagent and the GLDH enzyme activity present in each mL of the second reagent is from 15:1 to 1.5:1, more specifically from 10:1 to 2:1, even more specifically from 5:1 to 2:1, and even more specifically the ratio is about 3:1. In another embodiment, the amount of GLDH enzyme activity contained in the second reagent is from 1% to 30%, relative to the total GLDH enzyme activity provided by the reagents contained in the kit, and the amount is more specifically selected from any one of the following: 2% to 5%, 5% to 7%, 7% to 10%, 10% to 15%, 15% to 20%, 20% to 25% and 25% to 30%.

[0058] The above description of the embodiments of the first aspect also applies to the second aspect and other aspects. Therefore, the second aspect related to all other aspects and embodiments of the present disclosure is a method for providing a test kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample, the kit comprising two different aqueous reagents, the reagents comprising 2-oxoglutarate, glutamate dehydrogenase (=GLDH) capable of reacting NAD(P)H as a co-substrate, and NAD(P)H in an aqueous solution, the method comprising the following steps: preparing a first reagent having a pH capable of maintaining the enzymatic activity of GLDH by dissolving GLDH in an aqueous solution, preparing a second reagent by dissolving 2-oxoglutarate, GLDH, and NAD(P)H in an aqueous solution, and adjusting the pH of the second reagent to allow the enzymatic activity of GLDH in the second reagent to be maintained to convert ammonia, NAD(P)H, and 2-oxoglutarate, thereby allowing L-glutamate, NAD(P)H, and glutamate to be formed in the second reagent. + and H2O, providing the first reagent and the second reagent in separate containers, and combining the containers in a kit of parts, thereby providing an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

[0059] The third aspect related to all other aspects and embodiments of the present disclosure relates to use of the assay kit according to the first aspect or the assay kit obtained by implementing the method of the second aspect for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

[0060] In view of ammonia contamination caused by decomposition of co-substrates, another significant advantage of the technology introduced in this report is to extend the shelf life and improve the stability of reagents prepared as aqueous solutions and containing NAD(P)H. Therefore, the fourth aspect related to all other aspects and embodiments of the present disclosure relates to an assay kit according to the first aspect or an assay kit obtained by implementing the method of the second aspect, wherein the concentration of ammonia in the second reagent is about 1.5 μM or less, more specifically from about 0.01 μM to about 1.5 μM.

[0061] In a fifth aspect related to all other aspects and embodiments of the present disclosure, a mixture is provided, comprising (i) an aqueous liquid sample containing ammonia, and (ii) a second reagent of an assay kit, including an assay kit according to the first aspect or an assay kit obtained by implementing the method of the second aspect. In a specific example, the amount of ammonia from the second reagent plus the amount of ammonia present in the sample is about 1.5 μM or less.

[0062] As a sixth aspect related to all other aspects and embodiments of the present disclosure, an automated device capable of forming a mixture according to the fifth aspect is reported herein, wherein the device is combined with (i) an aqueous liquid sample in a sample container, and (ii) an assay kit according to the first aspect or an assay kit obtained by implementing the method of the second aspect. In one embodiment related to all other aspects and embodiments, the assay kit includes a reagent cartridge, wherein the reagent cartridge includes at least a first sealed compartment and a second sealed compartment, wherein the first compartment is a first container according to the first aspect given above, and therefore, the second compartment is a second container. Therefore, in one embodiment related to all aspects and embodiments provided herein, an assay kit of the first aspect is provided, wherein the first container and the second container are combined in any one of a retainer, a rack, and a cartridge, wherein the openings of the first container and the second container are closed and sealed. Therefore, any user can identify whether the solution is in its original state after manufacture. In a more specific embodiment, the first container and the second container are combined in a container with a Roche Diagnostics C platform C 311, C 501 / 502 and C 701 / 702 compatible In this embodiment, the reaction cartridge provides separate compartments for use as the first container and the second container as provided herein.

[0063] The following examples and drawings are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention.

[0064] Example 1

[0065] Diagnostic assay of ammonia in plasma samples using a prior art assay kit (assay name "NH3")

[0066] Roche for Roche / Hitachi analyzers is provided NH3 (ammonia) assay kit (including MODULAR P platform; assay kit purchased from Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 11877984216, with Cat. Nos. 20751995190, 20752401190, 20753009190 as calibrators).

[0067] Prior to use, R2 was prepared in three steps to dissolve a solid (i.e., anhydrous) preparation of NADPH into solution:

[0068] (1) Provide a vial 2a and a vial 2b, respectively, which are provided as components of an assay kit, and then reconstitute the contents of vial 2b by adding 0.5 mL of the reagent from vial 2a to vial 2b and mixing, and then incubate the mixture at room temperature for 10 minutes with occasional gentle agitation, thereby obtaining a first solution in vial 2b.

[0069] (2) Provide a vial 2 supplied as a component of the assay kit and vial 2a from step (1), and then reconstitute the contents of vial 2 by adding 4.5 mL of reagent from vial 2a to vial 2 and gently inverting to mix, thereby obtaining a second solution in vial 2.

[0070] (3) The first solution obtained in step (1) is provided to vial 2b, and the second solution obtained in step (2) is provided to vial 2, 150 μl of the first solution from vial 2b is added to the second solution in vial 2, and gently inverted to mix, to obtain a ready-to-use R2 working solution.

[0071] The working solution R1 provided as part of the kit contains triethanolamine buffer: 151 mmol / L, pH 8.6; 2-oxoglutaric acid: 16.6 mmol / L; ADP: ≥1.2 mmol / L; and additional preservatives.

[0072] The ready-to-use working solution R2 obtained as a result of step (3) detailed above comprises NADPH: ≥458 μmol / L; GLDH (beef liver; 25°C): ≥24.3 U / mL; triethanolamine buffer: 151 mmol / L, pH 8.6; 2-oxoglutaric acid: 16.6 mmol / L; ADP: ≥1.2 mmol / L; and additional preservatives.

[0073] Assay kits, specifically R1 and R2, were used on the Roche / Hitachi MODULAR P platform according to the manufacturer's instructions.

[0074] Example 2

[0075] Ammonia scavenging by adding different amounts of GLDH to R2

[0076] R2 working solution was prepared as described in Example 1. Aliquots were mixed with different amounts of GLDH

[0077] Example 3

[0078] Diagnostic determination of ammonia in plasma samples using a novel assay kit with enhanced NADPH stability (assay name "NH3L2")

[0079] Newly developed for Roche / Hitachi analyzers available NH3L2 (ammonia) assay kit (including cobas c platform c 311, c 501 / 502; the assay kit was constructed by the inventor, using Cat. No. 20751995190, 20752401190, 20753009190 as calibrators). The kit includes two different working solutions, namely R1 and R3, which are ready-to-use.

[0080] The working solution R1 provided as part of the kit contains BICINE (=N,N-bis(2-hydroxyethyl)-glycine) buffer: 300 mmol / L, pH 8.3; GLDH (microorganism): ≥16.7 μμkat / L; detergent; preservative.

[0081] Working solution R3 provided as part of the kit contains GLDH (microorganism): ≥5.0 μkat / L; 2-oxoglutarate: 78 mmol / L; NADPH: ≥1.3 mmol / L; stabilizer; non-reactive buffer.

[0082] according to FIG. 3A to FIG. 3C The instructions depicted in the assay kits were used on the Roche / Hitachi cobas c platform, specifically R1 and R3.

[0083] Example 4

[0084] Ammonia scavenging by adding different amounts of GLDH to R2

[0085] Table 1

[0086]

[0087]

[0088] The aqueous solution was freshly prepared, containing 2-oxoglutaric acid: 78 mmol / L; NADPH: ≥1.3 mmol / L; and a non-reactive buffer. Different amounts of GLDH were added to aliquots of the aqueous solution. One aliquot was additionally spiked with a known amount of ammonia. Different incubation conditions were applied to the individually prepared test solutions.

[0089] Table 1 summarizes the compositions and conditions. Figure 5 is a graphical representation of the results, i.e. the detected ammonia concentration (also called "c[NH3]") after each experiment is depicted by the height of each individual block in the 3D plot. The blocks depicted are along Figure 5The more the Y-axis given by the arrow in the figure extends (i.e. the higher the corresponding block in the figure), the higher the ammonia concentration detected in the corresponding experiment. Each block represents an experiment given in the numbered list in Table 1, and Figure 5 The numbered names of the individual blocks in correspond to the numbers [#] in Table 1.

[0090] Notably, by the presence of GLDH, the concentration of ammonia was reproducibly maintained in the range between 0.01 μM and 1.5 μM. Figure 5 The block in the graph associated with GLDH corresponds to values ​​from 0.02 uM to 1.3 μM.

[0091] At the end of the incubation at 35°C for experiments ##10, 15, 20, 25 and 30 (ie at the end of the 14 day interval), the remaining viable GLDH activity in the aqueous solution was determined. Table 2 summarizes the results.

[0092] Table 2

[0093]

[0094] It should be noted that the incubation conditions applied were designed to simulate the strains, and in particular the enzyme activities contained therein, being exposed to the reagents for a longer period of time under ambient conditions (e.g. room temperature). Thus, the data reported here clearly demonstrate the presence of a GLDH-based ammonia scavenging mechanism that affects the shelf life of the reagents.

[0095] Interestingly, even the lowest amount of GLDH (experiment #10) was able to successfully remove any ammonia formed during the incubation. When this is compared to the results for the same solution but without GLDH (experiment #5), it is clear that even under constrained conditions, i.e., conditions that are not optimal for GLDH activity and / or stability, it is possible to find conditions where the enzyme activity is sufficient to ensure efficient removal of ammonia generated by the decomposition of the co-substrate.

[0096] Since the cosubstrate was present at saturating concentrations, the consumption during the clearance process was found to be negligible, i.e., similar to the newly developed The performance of the NH3L2 (ammonia) assay kit is irrelevant.

[0097] Example 5

[0098] Stabilized working solution using NADH instead of NADPH

[0099] Working solution R3 as given in Example 2 and working solution R3' in which NADH replaced NADPH were prepared. Each solution was loaded into a cuvette and the extinction at or near the UV absorption maximum of NADH or NADPH was continuously measured. At a certain time point (after 15 minutes), an ammonia solution was added to the cuvette and the ammonia concentration in the corresponding working solution was adjusted to 100 μM. The extinction was further recorded. It was found that in both working solutions, the concentration of the co-substrate was reduced, which became apparent by the reduced extinction readings. It was concluded from these data that the mechanism of removing ammonia from the working solution (by GLDH enzyme activity) works in the presence of two co-substrates. Measurements were carried out at 10°C.

[0100] Figure 6 The extinction readings for NADPH (solid line) and NADH (dashed line) are depicted. The time point of addition of ammonia is indicated by the thin arrow, which also marks the onset of co-substrate consumption, indicated by the decreasing extinction.

[0101] It should be noted here that the amount of ammonia added was very high and was chosen at this concentration to show that even under suboptimal conditions, small amounts of GLDH are able to react ammonium and co-substrate to produce L-glutamate and water, thereby scavenging ammonia from solution.

Claims

1. A test kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample, the kit comprising 2-oxoglutarate, glutamate dehydrogenase (=GLDH) capable of reacting with NAD(P)H as a co-substrate, and NAD(P)H, The kit comprises a first container and a second container, wherein the first container comprises a first aqueous reagent having a first amount of GLDH, the first aqueous reagent having a pH capable of maintaining the enzymatic activity of GLDH, wherein the second container comprises a second aqueous reagent having NAD(P)H, 2-oxoglutaric acid, and a second amount of GLDH, wherein the GLDH enzyme activity per mL of the first aqueous reagent is higher than the GLDH enzyme activity per mL of the second aqueous reagent, and The pH of the second aqueous reagent is capable of maintaining the enzymatic activity of GLDH in the second aqueous reagent to convert ammonia, NAD(P)H and 2-oxoglutarate, thereby forming L-glutamate, NAD(P)H and 2-oxoglutarate in the second aqueous reagent. + and H2O.

2. The assay kit according to claim 1, wherein NAD(P)H is present only in the second aqueous reagent. 3 . The assay kit according to claim 1 , wherein the pH of the first aqueous reagent is from pH 7 to pH 9. 4 . 4 . The assay kit according to claim 1 , wherein the pH of the second aqueous reagent is from pH 8 to pH 11. 5 .

5. The assay kit according to any one of claims 1 and 2, wherein the ratio between the GLDH enzyme activity per mL of the first aqueous reagent and the GLDH enzyme activity per mL of the second aqueous reagent is from 15:1 to 1.5:

1.

6. The assay kit according to any one of claims 1 and 2, wherein the second aqueous reagent comprises GLDH enzyme activity in an amount of from 1% to 30% relative to the total GLDH enzyme activity provided by the reagents included in the kit.

7. The assay kit according to any one of claims 1 and 2, wherein the first aqueous reagent comprises a buffer selected from N,N-bis(2-hydroxyethyl)-glycine and triethanolamine.

8. The assay kit according to any one of claims 1 and 2, wherein the first aqueous reagent comprises a detergent.

9. A method for providing an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample, the kit comprising two different aqueous reagents, the reagents comprising 2-oxoglutarate, glutamate dehydrogenase (=GLDH) capable of reacting with NAD(P)H as a co-substrate, and NAD(P)H in aqueous solution, The method comprises the following steps: The first reagent having a pH capable of maintaining the enzymatic activity of GLDH is prepared by dissolving GLDH in an aqueous solution, The second reagent is prepared by dissolving 2-oxoglutarate, GLDH and NAD(P)H in an aqueous solution, and the pH of the second reagent is adjusted to allow the enzyme activity of GLDH in the second reagent to be maintained to convert ammonia, NAD(P)H and 2-oxoglutarate, thereby allowing L-glutamate, NAD(P)H and 2-oxoglutarate to be formed in the second reagent. + and H2O, providing the first reagent and the second reagent in separate containers, and combining the containers in a kit of parts, thereby providing an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

10. Use of the assay kit according to any one of claims 1 and 2 or the assay kit obtained by carrying out the method according to claim 9 for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

11. The assay kit according to any one of claims 1 and 2 or an assay kit obtained by carrying out the method according to claim 9, wherein the concentration of ammonia in the second aqueous reagent is 1.5 μM or less.

12. The assay kit according to claim 11, wherein the second aqueous reagent is obtained by the following steps: (a) dissolving 2-oxoglutarate, GLDH and NAD(P)H in an aqueous solution, and adjusting the pH of the second aqueous reagent to allow the enzyme activity of GLDH in the second aqueous reagent to be maintained to convert ammonia, NAD(P)H and 2-oxoglutarate, and then (b) incubating the aqueous solution obtained from step (a) at 35° C. for 14 days, The concentration of ammonia in the second aqueous reagent is from 0.02 μM to 1.3 μM.

13. The assay kit according to claim 11, wherein in the second aqueous reagent, ammonia released by decomposition of NAD(P)H is obtained by reacting ammonium ions, NAD(P)H and 2-oxoglutaric acid to form L-glutamic acid, NAD(P) + The GLDH enzyme activity and H2O are removed from the aqueous solution.

14. The assay kit according to claim 12, wherein in the second aqueous reagent, ammonia released by decomposition of NAD(P)H is obtained by reacting ammonium ions, NAD(P)H and 2-oxoglutaric acid to form L-glutamic acid, NAD(P) + The GLDH enzyme activity and H2O are removed from the aqueous solution.

15. An automated device capable of forming a mixture comprising (i) an aqueous liquid sample suspected of containing ammonia and (ii) the second aqueous reagent of the assay kit according to claim 13 or 14, wherein the device is combined with (i) an aqueous liquid sample in a sample container, and (ii) The kit according to any one of claims 1 and 2 or an assay kit obtained by carrying out the method according to claim 9.

16. The automated device of claim 15, wherein the sample in the mixture is a biological sample.

17. The automated device according to any one of claims 15 and 16, wherein the mixture further comprises the first aqueous reagent of the assay kit.

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