Stabilization of nadph or nadh in ammonia detection assays

Abstract

To provide an assay kit that improves stability of a reagent containing NAD(P)H and enhances shelf life, in an enzyme-based test for ammonia that can be used for analysis of plasma samples taken from patients in clinical settings.SOLUTION: In an exemplary reagent, ammonia released as a result of NAD(P)H decay is scavenged using an enzymatic reaction to convert the ammonia using GLDH, NAD(P)H and 2-oxoglutarate, thereby forming L-glutamate, NAD(P)+ and H2O in the NAD(P)H containing reagent.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure deals with the biochemistry of a reagent useful for the detection of ammonia in a liquid sample. Specifically, the present disclosure relates to technical improvements in an enzyme-based test for ammonia that can be used, among other uses, for the analysis of plasma samples taken from patients in a clinical setting. In this regard, the stability of reagents containing NAD(P)H is improved, resulting in an increased shelf life and detection of ammonia. In an exemplary reagent, ammonia released as a result of NAD(P)H decay is captured using an enzymatic reaction that converts ammonia using GLDH (glutamate dehydrogenase), NAD(P)H and 2-oxoglutarate, thereby providing L-glutamate, NAD(P)H in the NAD(P)H-containing reagent. + and H2O is formed [Background technology]

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

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

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

[0005] JP 03614967 B2 discloses the removal of ammonium from a sample using a combination including 2-oxoglutarate, GLDH, glucose-6-phosphate, glucose-6-phosphate dehydrogenase and NADPH.

[0006] Chenault KH & Whitesides GM Appl Biochem Biotechnol. 14 (1987) 147-197 report a different approach for regenerating the nicotinamide cofactor.

[0007] An exemplary embodiment of the Dafonseca-Wolheim modified method in the field of clinical chemistry and in vitro diagnostics comprises the cobas® NH3 (ammonia) assay kit for Roche / Hitachi analyzers for the quantitative measurement of ammonia in plasma (MODULAR P, cobas c platform; e.g., as a calibrator The kits in use include Roche catalog numbers 20751995190, 20752401190, 20753009190, and catalog number 11877984216). The kits in use include reagent containers containing first and second aqueous working solutions, also designated R1 and R2.

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

[0009] The aqueous solution must be kept under appropriate conditions to ensure the stability and shelf life of the reactive compounds involved. Specifically, GLDH becomes increasingly unstable as the pH increases. For diagnostic assay purposes, GLDH is not technically practical when stored under alkaline conditions above pH 10. However, a pH below pH 9 allows sufficient stability, shelf life and usefulness of GLDH. Thus, in the exemplary cobas® NH3 (ammonia) assay kit, the working solution R1 is an aqueous solution buffered to pH 8.3 (i.e., less than pH 9) that is provided by the manufacturer as a ready-to-use liquid reagent in the commercial assay kit.

[0010] In contrast, NADPH (and similarly NADH) is sufficiently stable in solution at pHs above pH 10. However, over time, NAD(P)H nevertheless exhibits decay in aqueous solutions even at pHs above pH 10. To address this instability, the cobas® NH3 (ammonia) assay kit is shipped with a dry component of R2, which contains NADPH. If maintained as a dry substance, NADPH remains stable. Prior to use, the R2 working solution is prepared by dissolving dry NADPH in an aqueous buffer, thereby forming an aqueous solution (R2) ready for subsequent (preferably immediate) consumption in the diagnostic assay workflow for measuring NH3 in a sample. As this requires extra manual labor, a stabilized ready-to-use reagent containing NADPH is desirable.

[0011] As a result of NADPH decay, ammonia is formed as a product. For this particular reason, there is a technical need to counteract NADPH decay in reagents for use in assays that measure and quantify ammonia in samples. Thus, over time, there is a risk that NH3 will accumulate in the R2 working solution due to NADPH decay, resulting in falsely elevated measurements.

[0012] The compositions, kits, methods and uses described in the present disclosure are designed to provide improvements in consideration of the above technical needs.Based on the teachings detailed herein, the assay for measuring ammonia in liquid samples can be advantageously improved.Surprisingly, the shelf life of NAD(P)H-containing aqueous reagent can be extended even under harsh conditions. Summary of the Invention

[0013] The present disclosure provides, as a first aspect, which relates to all other aspects and embodiments of the present disclosure, an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample, comprising first and second containers containing 2-oxoglutarate, glutamate dehydrogenase (=GLDH) capable of reacting NAD(P)H as a co-substrate, and NAD(P)H, the first container containing a first aqueous reagent comprising a first amount of GLDH, and the first The reagent has a pH capable of maintaining the enzymatic activity of GLDH, the second container contains a second aqueous reagent including NAD(P)H, 2-oxoglutarate, and a second amount of GLDH, the GLDH enzymatic activity per mL of the first reagent is greater than the GLDH enzymatic activity per mL in the second reagent, and 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 producing L-glutamate, NAD(P)H, and L-glutamate in the second reagent. + and a kit capable of producing HO is reported.

[0014] A second aspect herein, which is related to all other aspects and embodiments of the present disclosure, is a method of 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 containing 2-oxoglutarate, glutamate dehydrogenase (=GLDH) capable of reacting with 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 by dissolving GLDH in an aqueous solution having a pH capable of maintaining the enzymatic activity of GLDH, adjusting a second reagent by adjusting the pH of the second reagent to allow dissolving 2-oxoglutarate, GLDH, and NAD(P)H in the aqueous solution and maintaining the enzymatic activity of GLDH in the second reagent for conversion to ammonia, NAD(P)H, and 2-oxoglutarate, thereby reducing the amount of L-glutamate, NAD(P)H, and NAD(P)H in the second reagent. +and HO, providing the first and second reagents in separate containers, and assembling the containers into a kit of parts, thereby providing an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

[0015] Herein reported as a third aspect, related to all other aspects and embodiments of the present disclosure, is the use of an assay kit according to the first aspect or an assay kit resulting from carrying out the method of the second aspect, for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

[0016] Reported herein as a fourth aspect, related to all other aspects and embodiments of the present disclosure, is an assay kit according to the first aspect, or resulting from carrying out the method of the second aspect, wherein the concentration of ammonia in the second reagent is about 1.5 μM or less, more specifically about 0.01 μM to about 1.5 μM.

[0017] Reported herein as a fifth aspect, related to all other aspects and embodiments of the present disclosure, is a mixture comprising (i) an aqueous liquid sample suspected of 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 from carrying out the method of the second aspect.

[0018] Reported herein as a sixth aspect, related to all other aspects and embodiments of the present disclosure, is an automated apparatus capable of forming a mixture according to the fifth aspect, wherein the apparatus 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 resulting from performing the method of the second aspect. [Brief description of the drawings]

[0019] [Figure 1] Chemical formula of NADP+ and NADPH [Diagram 2]Experimental UV / vis spectra of NADP+ and NADPH reported in De Ruyck J. et al. Chem Phys Lett 450 (2007) 119-122. [Figure 3A] Description of working instructions for an improved diagnostic assay for ammonia detection and quantification in accordance with the teachings of this report. [Figure 3B] Description of working instructions for an improved diagnostic assay for ammonia detection and quantification in accordance with the teachings of this report. [Figure 3C] Description of working instructions for an improved diagnostic assay for ammonia detection and quantification in accordance with the teachings of this report. [Figure 4-1] Depiction of the molecular structures of the oxidized forms of the cosubstrates of GLDH: (A) NAD+, (B) NADP+, (C) 3-acetylpyridine-NAD+, (D) 3-acetylpyridine-NADP+, (E) 3-pyridinealdehyde-NAD+, (F) 3-pyridinealdehyde-NADP+, (G) thionicotinamide-NAD+, (H) thionicotinamide-NADP+. [Figure 4-2] Depiction of the molecular structures of the oxidized forms of the cosubstrates of GLDH: (A) NAD+, (B) NADP+, (C) 3-acetylpyridine-NAD+, (D) 3-acetylpyridine-NADP+, (E) 3-pyridinealdehyde-NAD+, (F) 3-pyridinealdehyde-NADP+, (G) thionicotinamide-NAD+, (H) thionicotinamide-NADP+. [Diagram 5] Graphical representation of the results of the experiments listed in Table 1 (see Example 4 and Table 1). [Figure 6] Comparison of (i) the original second reagent (R3) [solid line] working solution (containing NADPH) described in Example 2 and (ii) the modified working solution R3' (modified second reagent according to the present disclosure) in which NADPH is replaced with NADH [dotted line], for further details see Example 5. Absorbance readings of NADPH [solid line] and NADH [dotted line]. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0021] In this detailed description, reference to "one embodiment," "embodiment," or "in an embodiment" means that the referenced feature is included in at least one embodiment of the technology in all its aspects according to the present disclosure. Moreover, individual references to "one embodiment," "an embodiment," or "embodiments" do not necessarily refer to the same embodiment. However, such embodiments are not mutually exclusive unless otherwise stated and unless readily apparent to one of ordinary skill in the art. Thus, the technology in all its aspects according to the present disclosure may include any various combinations and / or integrations of the embodiments described herein.

[0022] The terms "a," "an," and "the" generally include plural referents unless the context clearly dictates otherwise. As used herein, "plurality" is understood to mean two or more. For example, plurality refers to at least two, three, four, five, or more. As used herein, unless otherwise stated or apparent from the context, the term "or" is understood to be inclusive.

[0023] It will be further understood that, as used herein, the terms "include" and / or "have" specify the presence of stated features, items, steps, operations, elements, and / or components, but do not exclude the presence or addition of at least one other feature, item, step, operation, element, component, and / or group thereof. Similarly, "with" also specifies the presence of stated features, etc.

[0024] As used herein, the terms "comprising," "contains," "containing," "includes," "including," "has," "having," or any other variations thereof, are intended to mean a non-exclusive inclusion. For example, a process, method, article, or apparatus that includes a list of features is not necessarily limited to only those features and may include other features not expressly recited or inherent to such process, method, article, or apparatus. In contrast, "consists of," "consisting of," or any other variation thereof, specifies an exclusive list of features. In particular, an exclusive list of a given feature is to be understood as representing a particular embodiment of the non-exclusive list of those features.

[0025] As used herein, unless expressly stated to the contrary, "or" refers to an inclusive or rather than an exclusive or. For example, condition A or B is satisfied by any one of the following: 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), or A and B are both true (or exist).

[0026] As used herein, "substantially," "relatively," "generally," "typically," and "approximately" are relative modifiers intended to indicate acceptable variations from the property so modified. They are not intended to be limited to the absolute value or property it modifies, but rather to approach or approximate such physical or functional property. As used herein, unless otherwise specified or clear from the context, the term "about" is understood to be within normal tolerances in the art, e.g., within 2 standard deviations of the mean. About can 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 stated value. Unless otherwise clear from the context, all numerical values ​​provided herein can be modified by the term about.

[0027] "Ammonia" is the well-known compound of nitrogen and hydrogen with the formula NH3. Ammonia can be dissolved in water, and aqueous solutions of ammonia are alkaline. Ammonia has a base constant pK of 4.76. b Therefore, in an aqueous solution, a certain amount of ammonia molecules react with water molecules to produce ammonium ions (= "ammonium") and hydroxyl ions. As long as ammonia is present in an aqueous solution, it is understood that ammonia is in reaction equilibrium with ammonium, unless otherwise specified.

[0028] 2-oxoglutarate (=α-ketoglutarate) is one of two ketone derivatives of glutaric acid. Its anion, 2-oxoglutarate (=α-ketoglutarate), is an important biological compound. It is a ketoacid produced by the deamination of glutamate and is an intermediate in the Krebs cycle.

[0029] Glutamate dehydrogenase (=GLDH) is an enzyme present in the mitochondria of most microorganisms and eukaryotes. GLDH activity can convert L-glutamate to α-ketoglutarate and vice versa. It is understood that all aspects and embodiments of this report relate to GLDH (EC 1.4.1.3) that can react with NAD(P)H as a co-substrate.

[0030] Nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ) are co-substrates for GLDH. They consist of an AMP (adenosine monophosphate) molecule covalently bound to a nicotinamide mononucleotide, and thus a dinucleotide. NADP + The presence of an additional phosphate group on carbon 2 of the ribose ring of AMP makes it easier to convert NAD + These molecules are diffusible cosubstrates involved in oxidation / reduction (=redox) reactions. + and NADPH + are the oxidized forms of the respective cosubstrates. When reduced, carbon 4 of the nicotinamide ring becomes a hydride (H ‐ )stomach ion, a proton and two electrons. NAD+ / NADP+ always undergoes a two-electron oxidation or reduction. NADH and NADPH are abbreviations for the reduced forms. For GLDH enzymatic activity, not only NADH and NADPH can serve as substrates, but also their functional analogues. Some of these are shown in Figure 4A-H. For the purposes of this report, it is stated herein that the teachings described herein in all aspects and embodiments can also be advantageously carried out with any functional analogue, provided that the particular GLDH in use is capable of reacting the reduced form of the respective functional analogue with 2-oxoglutarate in a redox reaction, thereby generating the oxidized form of the functional analogue.

[0031] For the purposes of this disclosure, an "assay kit" is understood as a composition of parts that are combined for the purpose of performing an assay. Typically, this 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 a material in the sample. Importantly, the meaning of "assay kit" refers to the composition of parts before use, which depletes one or more parts contained in the kit. In certain embodiments of containers that contain liquid reagents, it is understood that the container is closed and sealed, if necessary, before use. Before use of the container, open it and break the seal, if present. The aspects and embodiments reported in this disclosure relate to the assay kit as a kit of parts, comprising at least two parts, part A and part B. Other parts may be present in the assay kit according to the present disclosure. Each part typically comprises one or more components. When providing a kit of parts, it is necessary to avoid, if possible, combining components within the parts (components that may chemically react with each other to form undesired products), especially during storage of the kit of parts. Therefore, the kit of parts is typically provided in such a way that their reactive components are separated from each other, at least during storage, to avoid undesired reactions. Although the technical problem posed by the undesired reaction of NAD(P)H is specifically addressed, it is understood that all other components in the assay kit portion disclosed herein are in a suitable environment, for example (but not limited to) the reagents are enclosed in a casing (container) that protects against evaporation of the liquid, the enzymes and substrates are provided in a solvent configured to allow use according to the purpose of the assay kit, and other means accepted in the art. The assay kit also typically includes either packaging material and documentation containing information regarding the intended use of the assay kit and recommended or required conditions for storage before use of the kit. With regard to the packaging material, certain embodiments thereof allow the user to recognize the kit that has already been opened and therefore may not be in its original condition after manufacture, for example, by an inventive seal.

[0032] The findings reported in this disclosure relate, on the one hand, to a modification of the Kristen reaction by Da Fonseca-Wollheim. The original enzymatic method is improved by the addition of ADP to the reaction mixture, the use of NADPH instead of NADH to eliminate interference from the reaction of endogenous LDH with endogenous pyruvate, and the substitution of plasma for the deproteinized supernatant (Da Fonseca-Wollheim FZ Klin Chem Klin Biochem 11 (1973) 421-425). However, on the other hand, the findings of this disclosure relate more generally to the stabilization of aqueous solutions of NADPH and NADH, and specifically for the purpose of determining ammonia, with the associated detection assay being based on the enzymatic activity of glutamate dehydrogenase.

[0033] Typically, the detection reagent specific for ammonia is i) quantitatively reacting with the ammonium present in the sample (ammonia is therefore in reaction equilibrium with ammonium in aqueous solution); and ii) are selected and designed to be capable of simultaneously producing a stoichiometric amount of detectable reaction product that reflects the amount of ammonia reacted.

[0034] The long-established assay principle utilizes the following reaction [Reaction 1], which is catalyzed by glutamate dehydrogenase (GLDH). In aqueous solution, GLDH reacts with NH + and catalyzes the reductive amination of 2-oxoglutarate by NADPH, thereby producing glutamate and NADP + Form.

[0035] [Reaction 1] [ka]

[0036] The co-substrate in [Reaction 1] is NADPH, the reduced form of nicotinamide adenine dinucleotide phosphate (CAS number 53-59-8), and the corresponding oxidized form is NADP +Alternatively, the co-substrate can be NADH, the reduced form of nicotinamide adenine dinucleotide (CAS number 58-68-4), the corresponding oxidized form of which is NAD + An alternative enzymatic reaction is shown below in [Reaction 2].

[0037] [Reaction 2] [ka]

[0038] As already mentioned above, as long as ammonia is present in the aqueous solution, ammonia is in reaction equilibrium with ammonium, unless otherwise specified. For the purposes of this disclosure, either [Reaction 1] or [Reaction 2], either alone or in combination, is also referred to as the "detection reaction". Furthermore, either NADPH or NADHc is also referred to as the "reduced co-substrate". Furthermore, NADP + and NAD + Either of these is also referred to as an "oxidized cosubstrate."

[0039] Importantly, both detection reactions yield the respective oxidized cosubstrate in stoichiometric amounts, thus reflecting the amount of ammonium reacted. The cosubstrates absorb strongly in UV light, allowing the difference between the reduced and oxidized cosubstrates to be detected photometrically. Thus, the difference in absorption spectra between the oxidized and reduced forms of the cosubstrate makes it easy to measure this conversion in the enzyme assays of [Reaction 1] or [Reaction 2].

[0040] Thus, to great advantage, NADH and NADPH are used as co-substrates in numerous quantitative assays based on enzyme-catalyzed redox reactions, in particular in such assays for assessing the concentration of ammonia in liquid aqueous samples. For each enzyme-catalyzed redox reaction involving one ammonium ion, one NAD(P)H molecule is converted to NAD(P). +There is a stoichiometric relationship in which NADH and NADPH are converted into NAD(P) and NAD(P) at the same time. Spectrophotometric measurements allow quantitative detection of any change in the initial amount of NADH or NADPH initially present during the reaction. For [Reaction 1] and [Reaction 2], the spectrophotometric measurement of each oxidized form reflects the amount of ammonium (and ammonia) reacted, by detectably forming the respective oxidized forms as reaction products in stoichiometric amounts. The co-substrate and acceptor compound 2-glutamate is present in molar excess, and the chemical equilibrium is shifted towards the products (L-glutamate + NAD(P)). + As a result, all available ammonia is eventually converted, i.e., reacts quantitatively, thereby producing equimolar amounts of NAD, + or NAD + occurs .

[0041] NAD(P)H molecules in aqueous solutions are unstable, i.e., they tend to decay and release ammonia. Example 3 lists in Table 1 experiments Nos. 1, 3, 4 and 5, which show that ammonia release over time is indeed the case for aqueous solutions of NAD(P)H. The technical challenges associated with such ammonia-releasing decay of the auxiliary substrate are particularly evident when using an aqueous solution of NAD(P)H as a reagent in the detection of ammonia in a sample. The lower the ammonia concentration in the sample, the more serious the risk posed by the aqueous reagent containing NAD(P)H, considering the possibility of a falsely elevated quantitative measurement of ammonia.

[0042] However, as can be shown by the inventors and documented in this disclosure, even under less than suboptimal conditions (especially pH above 9 or even higher), a certain percentage of GLDH remains active in the presence of 2-oxoglutarate and NAD(P)H. The impact of this finding can be illustrated, in a non-limiting manner, by the Roche Diagnostics cobas® NH3 diagnostic assay. The cobas® NH3 assay of the present invention uses two different reagents, R1 and R2, which are mixed with a sample suspected of containing ammonia. R1 contains 2-oxoglutarate as a ready-made liquid reagent. Just before use, R2 is freshly prepared from solid materials and an aqueous solution, and after preparation is complete, it contains NADPH, GLDH and 2-oxoglutarate. Thus, currently available cobas® NH3 diagnostic assay kits do not contain two ready-to-use assay reagents, but only one. The kit contains an additional bottle with liquid contents and solid materials for preparing the R2 reagent manually before use.

[0043] Thus, a first aspect, which relates to all other aspects and embodiments of the present disclosure, is an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample, comprising first and second containers containing 2-oxoglutarate, glutamate dehydrogenase (=GLDH) capable of reacting with NAD(P)H as a co-substrate, and NAD(P)H, the first container containing a first aqueous reagent comprising a first amount of GLDH, the first reagent maintaining the enzymatic activity of GLDH. the second container contains a second aqueous reagent comprising NAD(P)H, 2-oxoglutarate, and a second amount of GLDH, the GLDH enzymatic activity per mL of the first reagent is greater than the GLDH enzymatic activity per mL in the second reagent, and 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 producing L-glutamate, NAD(P)H, and L-glutamate in the second reagent. + and HO.

[0044] It is important to understand that neither the second reagent nor the first reagent in the assay kit provided herein require a separate manual preparation step before use. Thus, in one embodiment of the assay kit, the kit can contain (more specifically comprise) only two containers containing two aqueous solutions, one of the two solutions containing one or more compounds selected from NAD(P)H, GLDH and 2-oxoglutarate. In one embodiment, NAD(P)H is present only in the second reagent.

[0045] In one embodiment, the pH of the first reagent is between pH 7 and pH 9, and more particularly between pH 8 and pH 9. A particular embodiment is the pH value of the first reagent, the particular value being selected from 8.5 to 8.9.

[0046] Regarding the second reagent in particular, the central finding on which this report is based is that GLDH can be used under conditions that would be technically unsuitable for use in scavenging ammonia from NAD(P)H-containing reagents, most of which are therefore favorable for the stability of NAD(P)H. The conditions of the NAD(P)H reagent can be selected such that it is more ammonia-free, i.e. has a reduced tendency to release ammonia. Furthermore, the presence of the GLDH enzyme renders the reagent ammonia-free. An important factor in this regard is the pH of the reagent, which is selected to be substantially alkaline. Thus, in an embodiment, the pH of the second reagent is between pH 8 and pH 11, and more particularly between pH 8 and pH 10, and even more particularly between pH 9 and pH 10.

[0047] Example 2 shows, in a non-limiting manner, that the newly developed cobas® NH3L2 R1 Reagent provides the majority of the GLDH enzyme activity used to detect ammonia in a sample. Note that the R1 Reagent does not contain 2-oxoglutarate. This is an example of an embodiment where 2-oxoglutarate is present only in the second reagent.

[0048] The newly developed second (R3) reagent contains a smaller portion of GLDH, but is sufficient to maintain the ammonia scavenging mechanism. Thus, in one embodiment, the ratio of GLDH enzyme activity per mL present in the first reagent to GLDH enzyme activity per mL present in the second reagent is 15:1 to 1.5:1, more specifically 10:1 to 2:1, even more specifically 5:1 to 2:1, even more specifically the ratio is about 3:1. In another embodiment, the second reagent contains an amount of GLDH enzyme activity form of 1% to 30% with respect to the total GLDH enzyme activity provided by the reagents included in the kit, the amount being more specifically selected from any of 2% to 5%, 5% to 7%, 7% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, and 25% to 30%.

[0049] The above description of the embodiment relating to the first aspect is equally applicable to the second aspect and other aspects. Thus, the second aspect, which is related to all other aspects and embodiments of the present disclosure, is 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 containing 2-oxoglutarate, glutamate dehydrogenase (=GLDH) capable of reacting with 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 by dissolving GLDH in an aqueous solution having a pH capable of maintaining the enzymatic activity of GLDH, adjusting the pH of the second reagent to allow 2-oxoglutarate, GLDH, and NAD(P)H to be dissolved in the aqueous solution, and maintaining the enzymatic activity of GLDH in the second reagent for conversion to ammonia, NAD(P)H, and 2-oxoglutarate, thereby reducing the amount of L-glutamate, NAD(P)H, and NAD(P)H in the second reagent. + and HO; providing the first and second reagents in separate containers; and assembling the containers into a kit of parts, thereby providing an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

[0050] A third aspect, which is related to all other aspects and embodiments of the present disclosure, relates to the use of an assay kit according to the first aspect or an assay kit resulting from carrying out the method of the second aspect, for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

[0051] Another notable advantage of the technology presented in this report is the increased shelf life and improved stability of the reagents produced as aqueous solutions and containing NAD(P)H, taking into account the contamination of ammonia due to the decomposition of the co-substrate.Therefore, a fourth aspect, related to all other aspects and embodiments of the present disclosure herein, relates to an assay kit according to the first aspect, or an assay kit resulting from carrying out the method of the second aspect, wherein the concentration of ammonia in the second reagent is about 1.5 μM or less, more specifically about 0.01 μM to about 1.5 μM.

[0052] A fifth aspect, related to all other aspects and embodiments of the present disclosure, is a process for producing ammonia by: providing a mixture comprising an aqueous liquid sample suspected of containing ammonia and (ii) a second reagent of an assay kit comprising an assay kit according to the first aspect or an assay kit obtained from carrying out the method of the second aspect. In a particular example, the amount of ammonia from the second reagent added to the amount of ammonia present in the sample is about 1.5 μM or less.

[0053] 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, which 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 resulting from carrying out the method of the second aspect. In an embodiment related to all other aspects and embodiments, the assay kit comprises a reagent cassette, which comprises at least a first and a second sealed compartment, the first compartment being a first container according to the above given first aspect, and the second compartment being thus a second container. Thus, in an embodiment related to all aspects and embodiments provided herein, an assay kit of the first aspect is provided, in which the first and second containers are combined in one of a holder, a rack and a cassette, and the openings of the first and second containers are closed and sealed. This allows a user to recognize whether the solution is in its original state after production. In a more specific embodiment, the first and second containers are combined in a cobas® reagent cassette that is compatible with any of the Roche Diagnostics cobas® c platforms c311, c501 / 502, and c701 / 702. In this embodiment, the reaction cassette provides separate compartments that function as the first and second containers provided herein. EXAMPLES

[0054] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit or scope of the invention.

[0055] Example 1 Diagnostic determination of ammonia in plasma samples using a prior art assay kit (assay name "NH3") The Roche cobas® NH3 (ammonia) assay kit for Roche / Hitachi analyzers (including the MODULAR P platform; Roche Diagnostics GmbH, Mannheim, Germany, catalog number 11877984216 with catalog numbers 20751995190, 20752401190, 20753009190 as calibrators) was provided.

[0056] Prior to use, R2 was prepared in three steps whereby a solid (i.e., water-free) preparation of NADPH was put into solution: (1) Providing one each of bottle 2a and bottle 2b supplied as part of an assay kit, then reconstituting the contents of bottle 2b by adding 0.5 mL of reagent from bottle 2a to bottle 2b and mixing, and then incubating the mixture at room temperature for 10 minutes with occasional gentle swirling, thereby obtaining a first solution in bottle 2b. (2) Provide one bottle 2 supplied as part of the assay kit and bottle 2a from step (1), then reconstitute the contents of bottle 2 by adding 4.5 mL of reagent from bottle 2a to bottle 2 and gently inverting to mix, thereby obtaining a second solution in bottle 2. (3) The first solution resulting from step (1) is provided in bottle 2b, the second solution resulting from step (2) is provided in bottle 2, and 150 μl of the first solution from bottle 2b is added to the second solution in bottle 2 and mixed by gentle inversion to obtain a ready-to-use R2 working solution.

[0057] Working solution R1 provided as part of the kit contained triethanolamine buffer: 151 mmol / L, pH 8.6; 2-oxoglutarate: 16.6 mmol / L; ADP: >= 1.2 mmol / L; and also a preservative.

[0058] The ready-to-use working solution R2 obtained as a result of step (3) detailed above contained NADPH: ≥ 458 μmol / L; GLDH (bovine liver; 25°C): ≥ 24.3 U / mL; triethanolamine buffer: 151 mmol / L, pH 8.6; 2-oxoglutarate: ≥ 16.6 mmol / L; ADP: ≥ 1.2 mmol / L; and also contained preservatives.

[0059] The assay kits, specifically R1 and R2, were used on the Roche / Hitachi MODULAR P platform according to the manufacturer's instructions.

[0060] Example 2 Ammonia scavenging by adding different amounts of GLDH to R2 R2 working solution was prepared as described in Example 1. Aliquots were mixed with different amounts of GLDH.

[0061] Example 3 Diagnostic determination of ammonia in plasma samples using a novel assay kit with enhanced stabilization of NADPH (assay name "NH3L2") A newly developed cobas® NH3L2 (ammonia) assay kit for Roche / Hitachi analyzers (including cobas c platform c 311, c501 / 502; assay kit constructed by the inventors using catalog numbers 20751995190, 20752401190, 20753009190 as calibrators) was prepared. The kit contains two different working solutions, R1 and R3, ready to use.

[0062] Working solution R1 provided as part of the kit contained BICINE (=N,N-bis(2-hydroxyethyl)-glycine) buffer: 300 mmol / L, pH 8.3; GLDH (microorganisms): ≧16.7 μkat / L; detergent; preservative.

[0063] Working solution R3 provided as part of the kit contained GLDH (microbial): ≧5.0 μkat / L; 2-oxoglutarate: 78 mmol / L; NADPH: ≧1.3 mmol / L; stabilizers; non-reactive buffer.

[0064] The assay kits, specifically R1 and R3, were used according to the instructions shown in Figures 3A-3C on the Roche / Hitachi cobas c platform.

[0065] Example 4 Ammonia scavenging by adding different amounts of GLDH to R2 [Table 1]

[0066] Aqueous solutions were freshly prepared and contained 2-oxoglutarate: 78 mmol / L; NADPH: ≧1.3 mmol / L; and non-reactive buffer. Different amounts of GLDH were added to aliquots of the aqueous solutions. One aliquot was additionally spiked with a known amount of ammonia. Different incubation conditions were applied to the individually prepared test solutions.

[0067] Table 1 summarizes the compositions and conditions. Figure 5 is a graphical representation of the results, i.e., the ammonia concentration (also referred to as "c[NH3]") detected after each experiment is indicated by the height of each individual block in the 3D diagram. The more the depicted block extends along the Y-axis given by the arrow in Figure 5 (i.e., the higher the height of each block in the diagram), the higher the ammonia concentration detected in the respective experiment. Each block represents an experiment given in the numbered list in Table 1, and the number designation given to the single blocks in Figure 5 corresponds to the numbering [#] in Table 1.

[0068] Remarkably, the presence of GLDH allowed the ammonia concentration to be reproducibly maintained in the range of 0.01 μM to 1.5 μM. The GLDH-related block in the diagram of Figure 5 corresponds to values ​​between 0.02 μM and 1.3 μM.

[0069] At the end of the 35° C. incubation in experiments no. 10, 15, 20, 25 and 30 (i.e., at the end of the 14-day intervals), the viable GLDH activity remaining in the aqueous solution was determined. The results are summarized in Table 2. [Table 2]

[0070] It should be noted that the applied incubation conditions were designed to mimic the strains provided for longer times at ambient conditions (e.g., room temperature) for the reagent, especially the enzymatic activities contained therein, so that the impact on the reagent shelf life caused by the presence of a GLDH-based scavenging mechanism for ammonia is made clear by the data reported here.

[0071] It is interesting to see that even the smallest amount of GLDH (experiment no. 10) was successful in removing the ammonia formed during the incubation period. Comparing this with the results for the same solution but in the absence of GLDH (experiment no. 5), it becomes clear that even under constraints, i.e. conditions that are not optimal for GLDH activity and / or stability, it is quite possible to find conditions in which the enzyme is active enough to ensure efficient removal of ammonia from the degradation of the co-substrate.

[0072] Since the co-substrate is present at saturating concentrations, its consumption in the capture process was found to be insignificant and therefore irrelevant to the performance of the newly developed cobas® NH3L2 (ammonia) assay kit.

[0073] Example 5 Stabilized working solution with NADH instead of NADPH A working solution R3 was prepared as shown in Example 2, and a working solution R3' was prepared in which NADH was replaced by NADPH. Each solution was filled into a cuvette and the absorbance was measured continuously at or near the UV absorption maximum of NADH or NADPH. At a certain time point (after 15 min), ammonia solution was added to the cuvette to adjust the ammonia concentration in each working solution to 100 μM. The absorbance was also recorded. In both working solutions, it was found that the concentration of the co-substrate was reduced, as evidenced by a decrease in the absorbance reading. From these data, it was concluded that the mechanism for scavenging ammonia from the working solution (due to GLDH enzyme activity) was functional in the presence of both co-substrates. Measurements were performed at 10°C.

[0074] Figure 6 shows absorbance readings for NADPH (solid line) and NADH (dotted line). The time of ammonia addition is indicated by the thin arrow indicating the onset of co-substrate consumption as indicated by absorbance.

[0075] It should be noted here that the amount of ammonia added was very large and this concentration was chosen to demonstrate that low amounts of GLDH, even under suboptimal conditions, are able to react ammonium and the co-substrate to produce L-glutamate and water, thereby scavenging ammonia from solution.

Claims

1. 1. An assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample, said kit comprising: 2-oxoglutarate; glutamate dehydrogenase (=GLDH) capable of reacting NAD(P)H as a co-substrate; and NAD(P)H; the kit comprising first and second containers; the first container contains a first aqueous reagent including a first amount of GLDH, the first reagent having a pH capable of maintaining the enzymatic activity of GLDH; the second container contains a second aqueous reagent comprising NAD(P)H, 2-oxoglutarate, and a second amount of GLDH; the GLDH enzyme activity per mL of the first reagent is greater than the GLDH enzyme activity per mL of the second reagent; and The pH of the second reagent can maintain the enzymatic activity of GLDH in the second reagent to convert ammonia, NAD(P)H and 2-oxoglutarate, thereby producing L-glutamate, NAD(P)H and 2-oxoglutarate in the second reagent. ＋ and H ２ An assay kit capable of forming O.

2. 2. The assay kit of claim 1, wherein NAD(P)H is present only in the second reagent.

3. 3. The assay kit of claim 1 or 2, wherein the pH of the first reagent is between pH 7 and pH 9, and more particularly between pH 8 and pH 9.

4. 4. The assay kit of claim 1, wherein the pH of the second reagent is between pH 8 and pH 11, and more particularly between pH 8 and pH 10, and even more particularly between pH 9 and pH 10.

5. 5. The assay kit of claim 1, wherein the ratio of GLDH enzyme activity per mL of the first reagent to GLDH enzyme activity per mL of the second reagent is between 15:1 and 1.5:1, more particularly between 10:1 and 2:1, even more particularly between 5:1 and 2:1, and even more particularly the ratio is about 3:

1.

6. 5. The assay kit of claim 1, wherein the second reagent contains an amount of GLDH enzyme activity form of 1% to 30% of the total GLDH enzyme activity provided by the reagents contained in the kit, the amount being more specifically selected from any of 2% to 5%, 5% to 7%, 7% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, and 25% to 30%.

7. The assay kit according to any one of claims 1 to 6, wherein the first reagent contains a buffer selected from N,N-bis(2-hydroxyethyl)-glycine and triethanolamine.

8. The assay kit of any one of claims 1 to 7, wherein the first reagent comprises a detergent.

9. A method for quantitatively determining the concentration of ammonia in an aqueous liquid sample, comprising providing an assay kit, the kit comprising two different aqueous reagents containing 2-oxoglutarate, glutamate dehydrogenase (=GLDH) capable of reacting with NAD(P)H as a co-substrate, and NAD(P)H in an aqueous solution, The method further comprising: preparing a first reagent by dissolving GLDH in an aqueous solution having a pH capable of maintaining the enzymatic activity of GLDH; The second reagent is prepared by dissolving 2-oxoglutarate, GLDH, and NAD(P)H in an aqueous solution, and adjusting the pH of the second reagent to allow maintaining the enzymatic activity of GLDH in the second reagent to convert ammonia, NAD(P)H, and 2-oxoglutarate, thereby reducing the amount of L-glutamate, NAD(P)H, and 2-oxoglutarate in the second reagent. ＋ and H ２ allowing for the formation of O; providing the first reagent and the second reagent in separate containers and combining the containers in a kit-of-parts to provide an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

10. Use of an assay kit according to any one of claims 1 to 8 or an assay kit obtained from carrying out the method according to claim 9 for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

11. 10. The assay kit according to any one of claims 1 to 8, or the assay kit obtained from carrying out the method according to claim 9, wherein the concentration of ammonia in the second reagent is less than or equal to about 1.5 μM, more particularly from about 0.01 μM to about 1.5 μM.

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

13. In the second reagent, ammonia released by the decomposition of NAD(P)H converts ammonium ions, NAD(P)H and 2-oxoglutarate into L-glutamate, NAD(P) ＋ and H ２ 13. The assay kit of claim 11 or 12, wherein the O is removed from the aqueous solution by GLDH enzyme activity reacting with O.

14. 14. A mixture comprising: (i) an aqueous liquid sample suspected of containing ammonia; and (ii) the second reagent of the assay kit of claim 13.

15. 15. The mixture of claim 14, wherein the sample is a biological sample, and more particularly plasma.

16. 16. The mixture of claim 14 or 15, wherein the mixture further comprises the first reagent of the assay kit.

17. 17. An automated device capable of forming the mixture of any one of claims 14 to 16, comprising: (i) an aqueous liquid sample in a sample container; and (ii) An automated device in combination with a kit according to any one of claims 1 to 8, or an assay kit resulting from carrying out the method according to claim 9.

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