Methods for detecting branched-chain alpha-keto acids or branched-chain amino acids

Abstract

The application relates to a buffer reagent, a kit, a branched-chain alpha-keto acid or a branched-chain amino acid detection method in the technical field of biological detection. The buffer reagent comprises 15-25 mM, pH 8.5-9.5 Tris-HCl buffer solution, 10-50 g / L trehalose, 0.1-1.5 g / L Proclin 300, 0.1-0.5 g / L Triton X-100, 0.1-1.5 g / L sodium salt of EDTA, 1-10 kU / L ascorbic acid oxidase and 0.5-3 g / L bovine serum albumin. The buffer reagent is suitable for preparation of a reagent containing leucine dehydrogenase, the reagent containing the buffer reagent and the leucine dehydrogenase and the kit prepared by using the reagent can effectively eliminate interference problems in the process of being used for branched-chain alpha-keto acid, branched-chain amino acid detection, and has good correlation with mass spectrometry.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a buffer reagent, a kit, and a detection method of branched-chain alpha-keto acid or branched-chain amino acid. BACKGROUND

[0002] Branched-chain amino acids (BCAAs) include leucine, isoleucine and valine, which have important physiological functions in the human body, such as participating in protein synthesis, regulating energy metabolism, enhancing immunity, etc. The changes in the content of branched-chain amino acids in serum and plasma are closely related to the occurrence and development of various diseases, such as cirrhosis, hepatic encephalopathy, diabetes, muscular dystrophy, etc. Therefore, accurately determining the content of branched-chain amino acids in serum and plasma has important clinical significance for early diagnosis, disease monitoring and treatment effect evaluation. The balance of branched-chain amino acids in the body is closely related to the occurrence and development of various diseases. Abnormal changes in the content of branched-chain amino acids in plasma can be used as an important biomarker for certain diseases. For example, abnormal metabolism of branched-chain amino acids is closely related to diseases such as diabetes, coronary heart disease, heart failure, etc., and the changes in their content can be used as a basis for disease prediction and prognosis evaluation. The content of branched-chain amino acids in normal human serum usually remains stable within a certain range, among which the normal range of leucine is about 0.8 mmol / L-1.2 mmol / L, the normal range of isoleucine is about 0.6 mmol / L-1.0 mmol / L, and the normal range of valine is about 0.9 mmol / L-1.5 mmol / L. These normal value ranges provide an important reference for clinical diagnosis and monitoring.

[0003] Branched-chain alpha-keto acids are intermediate metabolites of branched-chain amino acids (BCAAs) in the body. They are acted on by branched-chain alpha-keto acid dehydrogenase complex (BCKD) in cells, undergo transamination, dehydrogenation and further decomposition, and finally generate acetyl coenzyme A, enter the tricarboxylic acid cycle, and thus generate energy. Branched-chain alpha-keto acids have significant clinical significance in the medical field, and changes in their concentration can reflect the metabolic state of branched-chain amino acids in the body and are closely related to various diseases. For example, abnormal levels of branched-chain alpha-keto acids may be related to the occurrence and development of metabolic diseases, neurodegenerative diseases and certain cancers. Therefore, monitoring the levels of these compounds can provide valuable information for disease diagnosis, treatment monitoring and prognosis evaluation.

[0004] Currently, branched-chain alpha-keto acid detection mainly relies on high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), etc. Common branched-chain amino acid detection methods include liquid chromatography (HPLC), ion chromatography, spectrophotometry, mass spectrometry (MS), etc. However, the current detection methods have many shortcomings, such as complex operation. Although some simple methods have been proposed, they still face the problem of interference elimination and have poor correlation with mass spectrometry, making it difficult to promote the widespread clinical application of branched-chain alpha-keto acids and branched-chain amino acids.

[0005] Therefore, the present application is provided. SUMMARY

[0006] One or more embodiments of the present application provide a buffer reagent, a kit, a branched-chain alpha-keto acid or a branched-chain amino acid detection method. The technical solutions include the following:

[0007] One or more embodiments of the present application provide a buffer reagent, which comprises 15-25 mM, pH 8.5-9.5 Tris-HCl buffer, and 10-50 g / L trehalose, 0.1-1.5 g / L Proclin 300, 0.1-0.5 g / L Triton X-100, 0.1-1.5 g / L EDTA sodium salt, 1-10 kU / L ascorbate oxidase and 0.5-3 g / L bovine serum albumin.

[0008] In some embodiments of the present application, the buffer reagent comprises 18-22 mM, pH 8.5-9.5 Tris-HCl buffer, and 45-50 g / L trehalose, 0.8-1.5 g / L Proclin 300, 0.15-0.3 g / L Triton X-100, 0.15-0.25 g / L EDTA sodium salt, 1-5 kU / L ascorbate oxidase and 1-2 g / L bovine serum albumin.

[0009] One or more embodiments of the present application provide a kit, which comprises the buffer reagent.

[0010] In some embodiments of the present application, the kit further comprises one or more of leucine dehydrogenase, reduced coenzyme I, ammonium salt and oxidized coenzyme I.

[0011] In some embodiments of the present application, the amino acid sequence of the leucine dehydrogenase is shown in SEQ ID NO. 1.

[0012] In some embodiments of the present application, the kit comprises reagent 1 and reagent 2;

[0013] The reagent 1 comprises the leucine dehydrogenase and the buffer reagent, and comprises or does not comprise the reduced form of coenzyme I;

[0014] In the case that the reagent 1 comprises the reduced form of coenzyme I, the reagent 2 comprises the ammonium salt;

[0015] In the case that the reagent 1 does not comprise the reduced form of coenzyme I, the reagent 2 comprises the oxidized form of coenzyme I.

[0016] In some embodiments of the present application, the ammonium salt comprises ammonium sulfate.

[0017] In some embodiments of the present application, the kit satisfies one or more of the following conditions:

[0018] (1) the amount of the leucine dehydrogenase in the reagent 1 is 5 kU / L-20 kU / L;

[0019] (2) the amount of the reduced form of coenzyme I in the reagent 1 is 1 g / L-2 g / L;

[0020] (3) the amount of the ammonium salt in the reagent 2 is 10 g / L-30 g / L; and,

[0021] (4) the amount of the oxidized form of coenzyme I in the reagent 2 is 20 g / L-30 g / L.

[0022] In some embodiments of the present application, the reagent 2 further comprises 0.1 g / L-1.5 g / L of Proclin 300, 0.1 g / L-1.5 g / L of sodium EDTA, and 45 mM-55 mM, pH 4.0-5.0 glycine buffer.

[0023] One or more embodiments of the present application provide a detection method of branched chain amino acids or branched chain α-keto acids, which uses the kit to detect a sample to be tested.

[0024] In some embodiments of the present application, the detection method satisfies one or more of the following conditions:

[0025] 1) the detection method comprises ultraviolet-visible spectrophotometry; and,

[0026] 2) during the detection process, the volume ratio of the sample to be tested, the reagent 1 and the reagent 2 is (10-25):(85-95):(25-35).

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The present application provides a specific formula of a buffer reagent, which is suitable for the preparation of a reagent containing leucine dehydrogenase (especially the leucine dehydrogenase shown in SEQ ID NO. 1), the reagent containing the buffer reagent and the leucine dehydrogenase, and the kit prepared by the reagent can effectively eliminate interference problems in the process of detecting branched-chain alpha-keto acids and branched-chain amino acids, and has good correlation with mass spectrometry, good stability, and is conducive to promoting the wide popularization of branched-chain alpha-keto acid and branched-chain amino acid detection in clinical practice. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0030] Figure 1 Reaction curve of branched-chain alpha-keto acid concentration 200 μM in Example 1;

[0031] Figure 2 Sample correlation comparison of the detection method in Example 1 and mass spectrometry;

[0032] Figure 3 Reaction curve of branched-chain amino acid concentration 150 μM in Example 2;

[0033] Figure 4 Sample correlation comparison of the detection method in Example 2 and mass spectrometry;

[0034] Figure 5 Reaction curve of 200 μM alpha-ketoisocaproic acid in Example 3;

[0035] Figure 6 Verification result of the stability of the detection kit after opening in Example 3;

[0036] Figure 7 Correlation comparison of the detection method in Example 3 and mass spectrometry;

[0037] Figure 8 Reaction curve of 200 μM alpha-ketoisocaproic acid in Example 4;

[0038] Figure 9 Correlation comparison of the detection method in Example 4 and mass spectrometry;

[0039] Figure 10 To compare the detection method in Example 5 with the correlation of mass spectrometry;

[0040] Figure 11 To compare the detection method in Example 6 with the correlation of mass spectrometry;

[0041] Figure 12 To compare the detection method in Comparative Example 1 with the correlation of mass spectrometry;

[0042] Figure 13 To compare the detection method in Comparative Example 2 with the correlation of mass spectrometry. DETAILED DESCRIPTION

[0043] The present application will be further described with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to explain the present application and not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the disclosure of the present application more thoroughly and comprehensively understood. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the present application, and the equivalent forms obtained thereby also fall within the scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing embodiments and examples only and is not intended to be limiting of the present application.

[0045] Terminology

[0046] Unless otherwise indicated or unless contradicted by context, the terms or phrases used herein have the following meanings:

[0047] The selection range of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, which includes any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0048] In the present application, "multiple", "various", "multiple times", "multiple" and the like refer to greater than or equal to 2 in number, unless otherwise specified. For example, "one or more" means one or greater than or equal to two.

[0049] As used herein, "combinations thereof", "any combination thereof", "any combination manner thereof" and the like include all suitable combination manners of any two or more of the listed items.

[0050] As used herein, "suitable", "suitable", "any suitable manner" and the like are subject to the ability to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0051] As used herein, "preferably", "better", "better", "as appropriate" only describe the implementation manner or embodiment with better effect, and it should be understood that it does not constitute a limitation on the protection scope of the present application.

[0052] In the present application, "further", "more further", "in particular" and the like are used to describe the purpose, indicating the difference in content, but should not be understood as a limitation on the protection scope of the present application.

[0053] In the present application, "optionally", "optional", "optional" means optional, i.e. selected from two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified, and there is no contradictory relationship or mutual restriction, each "optional" is independent.

[0054] In the present application, the terms "first", "second", "third", "fourth" and the like in the "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are used only for descriptive purposes, and cannot be construed as indicating or implying relative importance or quantity, nor can they be construed as implicitly indicating the importance or quantity of the technical features indicated. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration and description, and should be understood as not constituting a closed limitation on the quantity.

[0055] In the present application, among the technical features described in an open manner, a closed technical solution composed of the listed features is also included, as well as an open technical solution containing the listed features.

[0056] In the present application, with respect to a numerical interval (i.e. a numerical range), if no special instructions are given, the optional numerical distribution within the above-mentioned numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range, as well as every numerical value between the two numerical end points. If no special instructions are given, when the numerical interval only points to the integers within the numerical interval, including the two end point integers of the numerical range and every integer between the two end points, in this document, it is equivalent to directly listing every integer, for example, t is an integer selected from 1-10, which means that t is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this document should be understood to include any and all sub-ranges encompassed therein.

[0057] In the present application, the temperature parameter, if not specifically limited, allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. It is allowed to fluctuate within the range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C.

[0058] In the present application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass volume percentage.

[0059] All the documents mentioned in the present application are cited in the present application as references, as if each document is cited as a reference individually. The cited documents are cited in the present application in their entirety, in their entirety purpose, unless and to the extent that the application purpose and / or technical scheme of the present application is conflicted. When the present application refers to the cited documents, the definition of the related technical features, terms, names, phrases, etc. in the cited documents are cited in the present application. When the present application refers to the cited documents, the examples, preferred modes of the cited related technical features can be cited in the present application as references, but limited to the implementation of the present application. It should be understood that when the cited content is conflicted with the description in the present application, the present application is correct or is amended according to the description in the present application adaptively.

[0060] In a first aspect, the present application provides a buffer reagent, which comprises 15-25 mM Tris-HCl buffer solution with pH 8.5-9.5, and 10-50 g / L trehalose, 0.1-1.5 g / L Proclin 300, 0.1-0.5 g / L Triton X-100, 0.1-1.5 g / L sodium salt of EDTA, 1-10 kU / L ascorbate oxidase and 0.5-3 g / L bovine serum albumin.

[0061] In the buffer reagent of the present application, the concentration of the Tris-HCl buffer solution is for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mM, and the pH is for example 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5.

[0062] The concentration of trehalose in the buffer reagent of the present application is, for example, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 g / L. The concentration of Proclin 300 in the buffer reagent of the present application is, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5 g / L. The concentration of Triton X-100 in the buffer reagent of the present application is, for example, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5 g / L. The concentration of sodium EDTA in the buffer reagent of the present application is, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5 g / L. The concentration of ascorate oxidase in the buffer reagent of the present application is, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 kU / L. The concentration of bovine serum albumin in the buffer reagent of the present application is, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3 g / L.

[0063] In some examples of the present application, the buffer reagent comprises 18-22 mM Tris-HCl buffer at pH 8.5-9.5, and 45-50 g / L trehalose, 0.8-1.5 g / L Proclin 300, 0.15-0.3 g / L Triton X-100, 0.15-0.25 g / L sodium EDTA, 1-5 kU / L ascorate oxidase, and 1-2 g / L bovine serum albumin.

[0064] The concentration of each component in the buffer reagent of the present application refers to the working concentration, i.e. the concentration in use. On the basis of meeting the working concentration, the buffer reagent of the present application is not particularly limited, and can be ready-to-use or ready-to-use.

[0065] In a second aspect of the embodiments of the present application, a kit is provided, which comprises the buffer reagent.

[0066] The kit of the present application can also include other reagents or devices required for detection. In some examples of the present application, the kit further comprises one or more of leucine dehydrogenase, reduced coenzyme I, ammonium salt, and oxidized coenzyme I.

[0067] In some examples of the present application, the amino acid sequence of the leucine dehydrogenase is as shown in SEQ ID NO. 1:

[0068] MGKNESSTNATNTKQWRDETKGFRDEAKRFKNTAGHHHHHHHHGSGMKIFDYMEKYDYEQLVMCQDKESGLKAIICIHVTTLGPALGGMRMWTYASEEEAIEDALRLGRGMTYKNAAAGLNLGGGKTVIIGDPRRDKNEAMFRALGRFIQGLNGRYITAEDVGTTVEFMDIIHEETRYVTGVSPAFGSSGNPSPVTAYGVYRGMKAAAKEAFGDDSLEGKVVAVQGVGHVAYELCKHLHNEGAKLIVTDINKENADRAVQEFGAEFVHPDKIYDVECDIFAPCALGAIINDETIERLKCKVVAGSANNQLKEERHGKMLEEKGIVYAPDYVINAGGVINVADELLGYNRERAMKKVEGIYDKILKVFEIAKRDGIPSYLAADRMAEERIEMMRKTRSTFLQDQRNLINFNNKDD (N-terminal to C-terminal);

[0069] Alternatively, the amino acid sequence of the leucine dehydrogenase has at least 80% (at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%) identity to SEQ ID NO. 1.

[0070] "Identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of identity between two sequences is the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if six of ten positions in two sequences are matched or homologous when the two sequences are optimally aligned, the two sequences are 60% homologous; if 95 of 100 positions in two sequences are matched or homologous, the two sequences are 95% homologous. Typically, the comparison is made when two sequences are aligned to give the maximum percent identity. The comparison can be made, for example, by the BLAST algorithm, in which the parameters of the algorithm are selected to give the maximum match between the sequences over their entire length.

[0071] The leucine dehydrogenase shown in SEQ ID NO. 1 (i.e., the mutant) has D122F and K89R relative to the wild-type leucine dehydrogenase, has DD added at the C-terminus, and has a tag that facilitates soluble expression and purification added at the N-terminus. As a result of these modifications, the mutant has significantly improved stability, reaction rate, and yield relative to the wild-type leucine dehydrogenase, as shown below.

[0072] In terms of stability, the mutant has 86% residual enzyme activity after incubation at 40°C for 6 hours and 66% residual enzyme activity after incubation at 55°C for 1 hour, while the wild-type has only 38% and 13% residual enzyme activity under the same conditions, respectively.

[0073] In terms of reaction rate, the mutant has significantly improved reaction rate, with a reaction rate of 0.28 ΔA / min, while the wild-type has a reaction rate of only 0.0352, which makes the mutant more advantageous in terms of speed in clinical detection.

[0074] In terms of yield, the mutant can be expressed in E. coli, and the yield of fermentation in a shake flask is more than 100 mg / L, while the yield of the wild-type is only about 14 mg / L, which makes it possible to mass-produce the mutant in industry.

[0075] The mutant is stable in the above-mentioned buffer reagents, and the reagent / kit containing the mutant prepared using the above-mentioned buffer reagents can effectively eliminate interference problems during detection of branched-chain α-keto acids and branched-chain amino acids, and has good correlation with mass spectrometry, which is conducive to promoting the widespread popularity of branched-chain α-keto acid and branched-chain amino acid detection in clinical practice.

[0076] In some examples of the present application, the kit comprises reagent 1 and reagent 2.

[0077] The reagent 1 includes the leucine dehydrogenase and the buffer reagent, and includes or does not include the reduced coenzyme I;

[0078] In the case where the reagent 1 includes the reduced coenzyme I, the reagent 2 includes the ammonium salt.

[0079] In the case where the reagent 1 does not include the reduced coenzyme I, the reagent 2 includes the oxidized coenzyme I.

[0080] In some examples of the present application, the ammonium salt includes ammonium sulfate.

[0081] In some examples of the present application, the kit satisfies one or more of the following conditions:

[0082] (1) The amount of the leucine dehydrogenase in the reagent 1 is 5 kU / L to 20 kU / L (for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 kU / L).

[0083] (2) The amount of the reduced coenzyme I in the reagent 1 is 1 g / L to 2 g / L (for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 g / L).

[0084] (3) The amount of the ammonium salt in the reagent 2 is 10 g / L to 30 g / L (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 g / L); and,

[0085] (4) The amount of the oxidized coenzyme I in the reagent 2 is 20 g / L to 30 g / L (for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 g / L).

[0086] In some examples of the present application, the reagent 2 further comprises 0.1 g / L-1.5 g / L of Proclin 300, 0.1 g / L-1.5 g / L of sodium salt of EDTA, and 45 mM-55 mM of glycine buffer, pH 4.0-5.0. In the reagent 2, the concentration of Proclin 300 is, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5 g / L; the concentration of sodium salt of EDTA is, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5 g / L; and the concentration of glycine buffer is, for example, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 mM, and the pH is, for example, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.

[0087] In a third aspect, the present application provides a branched chain amino acid or branched chain alpha-keto acid detection method, which uses the reagent kit to detect the sample to be tested.

[0088] In some examples of the present application, the detection method meets one or more of the following conditions:

[0089] 1) the detection method comprises ultraviolet-visible spectrophotometry; and,

[0090] 2) during the detection process, the volume ratio of the sample to be tested, the reagent 1 and the reagent 2 is (10-25):(85-95):(25-35), for example, 10:85:25, 10:85:30, 10:85:35, 20:85:25, 20:85:30, 20:85:35, 25:85:25, 25:85:30, 25:85:35, 10:95:25, 10:95:30, 10:95:35, 20:95:25, 20:95:30, 20:95:35, 25:95:25, 25:95:30, 25:95:35, 10:90:25, 10:90:30, 10:90:35, 20:90:25, 20:90:30, 20:90:35, 25:90:25, 25:90:30, 25:90:35.

[0091] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate but not to limit the scope of the present application. The experimental methods in the following examples without specific conditions are preferred to refer to the guidance given in the present application, and can also be carried out according to the experimental manual or conventional conditions in the art, or according to the conditions suggested by the manufacturer, or according to the experimental methods known in the art.

[0092] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range if not specifically stated. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0093] Bovine Serum Albumin (brand: Proliant, product name: Bovine Serum Albumin New Zealand Manufactured Standard Grade pH 7.0, item number: 68100); ascorbate oxidase (manufacturer: Asahi KASEI, item number: T-53); leucine dehydrogenase (amino acid sequence shown in SEQ ID NO. 1); reduced coenzyme I (manufacturer: Roche, item number: 10004642103); oxidized coenzyme I (manufacturer: Roche, item number: 10004626103).

[0094] Example 1

[0095] The present example provides a branched-chain alpha-keto acid detection kit and a detection method. It includes the following technical solutions:

[0096] 1. Detection kit

[0097] The branched-chain alpha-keto acid detection kit includes reagent 1 and reagent 2, wherein:

[0098] The composition of reagent 1 is as follows: 20mM, pH=9.0 Tris-HCl buffer, trehalose 50g / L, EDTA 2Na 0.2g / L, Triton X-100 0.2g / L, bovine serum albumin 1g / L, ascorbate oxidase 2kU / L, leucine dehydrogenase 10kU / L, Proclin 300 1g / L, reduced coenzyme I 1g / L.

[0099] The composition of reagent 2 is as follows: 50mM pH=4.0 glycine buffer, EDTA 2Na 0.5g / L, ammonium sulfate 20g / L, Proclin 300 1g / L.

[0100] 2. Detection method

[0101] 2.1 Detection process

[0102] The reaction temperature is 37°C, the reaction time is 5 minutes, the test main wavelength is 340 nm, the sample to reagent ratio is sample: reagent 1: reagent 2 = 20 μL: 90 μL: 30 μL, and the reaction is a reduction reaction. The sample and reagent 1 are first added and mixed automatically inside the automatic biochemical analyzer, and the absorbance value at the main wavelength is detected and recorded. After 3 minutes, reagent 2 is added and mixed automatically inside the automatic biochemical analyzer, and the absorbance value at the main wavelength is recorded after 5 minutes. According to the end point method, the content of branched chain amino acid in the sample is calculated by comparing the corresponding standard curve. The reaction curve of the test sample with a branched chain alpha-keto acid (such as alpha-ketoisocaproic acid) concentration of 200 μM is shown in FIG. 1. Figure 1

[0103] The principle of detecting branched chain alpha-keto acid is as follows, taking alpha-ketoisocaproic acid as an example:

[0104] Alpha-ketoisocaproic acid (KIC) undergoes reductive amination reaction in the presence of reduced coenzyme I and ammonium ion under the catalysis of leucine dehydrogenase (LeuDH), and alpha-ketoisocaproic acid is reduced to L-leucine, while reduced coenzyme I is oxidized to oxidized coenzyme I. Reduced coenzyme I has a characteristic absorption peak at 340 nm, and its consumption rate is linearly positively correlated with the concentration of alpha-ketoisocaproic acid. The reaction equation is as follows:

[0105]

[0106] 2.2 Long-term stability verification

[0107] The reagent kit of this example has good stability, and the long-term stability experiment of the liquid reagent shows that it can be stored for 1 year at 2-8°C. The verification process and results are as follows.

[0108] (1) Sample preparation

[0109] Detection reagent: reagent 1 and reagent 2 under item 1.

[0110] Calibration: matched calibration (the concentration gradient of alpha-ketoisocaproic acid is 0 μM, 50 μM, 100 μM, 200 μM, and 400 μM, respectively).

[0111] (2) Storage condition: after sealing, the detection reagent and calibration are stored in a constant temperature environment at 2-8°C to simulate the actual product storage condition.

[0112] (3) Detection time node: 0th month (initial value), 1st month, 3rd month, 5th month, 7th month, 9th month, and 12th month.

[0113] ​(4) Detection parameters: The absorbance value (OD value) of each calibration sample point was measured using the fully automatic biochemical analyzer IBC 900, and each concentration point was repeated 3 times, and the average value was recorded, as shown in Table 1 below.

[0114] Table 1

[0115]

[0116] (5) Stability determination criteria

[0117] Taking the absorbance value measured at the 0th month (initial OD value) as the reference, the relative deviation (%) of the data at each subsequent time point (current OD value) and the initial OD value was calculated, and the formula was:

[0118]

[0119] Stability requirements: The deviation of all calibration sample points at each time point should be ≤10%.

[0120] The relative deviation analysis is shown in Table 2.

[0121] Table 2, Relative Deviation Analysis

[0122]

[0123] (6) Conclusion

[0124] Under the storage condition of 2-8°C, the branched-chain alpha-keto acid detection reagent and calibrators showed excellent stability within 12 months. The absorbance deviation of all calibrators (0-400 μM) was ≤6.04%, which was significantly lower than the preset threshold of 10%. The highest deviation value appeared at the 1st month of the 50 μM calibrator (6.04%), which still met the stability standard.

[0125] 2.3 Stability verification after opening (i.e., opening the cap)

[0126] After the reagents in the detection kit were calibrated, they were placed in the reagent compartment, and the fixed quality control was regularly detected to verify the stability performance of the kit during use. The verification process and verification results are as follows.

[0127] (1) After opening the reagent, it was placed in the reagent compartment of the IBC 900 biochemical analyzer to simulate the actual use scene in the hospital, and the following conditions were maintained: the temperature of the reagent compartment was controlled at 2-8°C, and the humidity was controlled at 50±10% (to avoid the performance fluctuation caused by reagent moisture absorption or volatilization).

[0128] (2) Biochemical quality control is a substance used to monitor the quality of the detection process, and its matrix and concentration level is close to that of clinical samples. It is used to evaluate the stability and reliability of the analysis system (instrument, reagent, operation) by regular testing. The branched-chain alpha-keto acid high-value (alpha-ketoisocaproic acid concentration of 100 μM) and low-value (alpha-ketoisocaproic acid concentration of 50 μM) quality control provided in the present application are used to test the bottle opening stability of the reagent.

[0129] (3) Test procedure

[0130] Initial calibration: calibrate the reagent on the day of opening (day 1) and determine the baseline value of the quality control;

[0131] Daily testing: continuously determine the concentration of the quality control and calculate the deviation from the results of day 1;

[0132] Stability criteria: absolute value of deviation ≤10% is determined to be stable.

[0133] The bottle opening stability results of the present example are shown in Table 3 below:

[0134] Table 3

[0135]

[0136] (4) Conclusion

[0137] After opening the reagent, it is placed in the reagent compartment of the biochemical analyzer. Under the conditions of 2-8℃ and humidity of 50±10%, the performance is stable and meets the clinical detection requirements.

[0138] 2.4 Correlation verification

[0139] The sample was tested using the detection method of the present example, and compared with mass spectrometry. The sample correlation is good, as shown in Table 4 and Figure 2 .

[0140] Table 4 (unit: μM)

[0141]

[0142] 50 clinical serum samples were tested using the detection kit and detection method of the present example. The sample has mass spectrometry results in the hospital. The correlation R 2 of the detection method of the present example and mass spectrometry is 0.981, and the correlation is good, as shown in Table 4 and Figure 2 above. All are the total amount of branched-chain alpha-keto acid in the sample. The mass spectrometry method is mentioned in Example 1 of the document with application number CN202410918742.6.

[0143] 2.5 Specificity verification

[0144] (1) Interfering substances

[0145] Non-branched alpha-keto acid: represented by pyruvic acid (alpha-ketopropionic acid), its chemical structure is similar to branched alpha-keto acid (such as alpha-ketoisovaleric acid), which is easy to compete with the substrate or chromogenic agent in enzymatic reaction;

[0146] Abnormal metabolic keto acid: represented by beta-ketoisocaproic acid, commonly found in samples of patients with genetic metabolic diseases (such as maple syrup urine disease), which coexists with target substances (such as alpha-ketoisocaproic acid) due to disorders of branched chain amino acid metabolism.

[0147] (2) Verification method

[0148] Prepare the same concentration (250 μM) of interferent solution (pyruvic acid, beta-ketoisocaproic acid) as the target substance (branched alpha-keto acid: alpha-ketoisocaproic acid); use IBC900 biochemical instrument to test the solution concentration, calculate the recovery rate and interference rate, the calculation formula is as follows: interference rate = test concentration / theoretical concentration * 100%; recovery rate = test concentration / theoretical concentration * 100%.

[0149] The data are shown in Table 5 below:

[0150] Table 5 (unit: μM)

[0151]

[0152] The average measured concentration of alpha-ketoisocaproic acid (target substance) is 244.6 μM (theoretical concentration 250 μM), and the recovery rate is 97.8%, indicating that the method has high detection accuracy. Pyruvic acid (non-branched alpha-keto acid): the average measured concentration is 6.9 μM, and the interference rate is 2.76%; beta-ketoisocaproic acid (abnormal metabolic keto acid): the average measured concentration is 9.7 μM, and the interference rate is 3.88%.

[0153] (3) Conclusion

[0154] The interference rates of the two types of interferents are significantly lower than the clinical detection threshold of 5%, which proves that the method has excellent anti-interference ability for structural analogues and metabolic related interferents.

[0155] 2.6 Sensitivity verification

[0156] By diluting the target substance (alpha-ketoisocaproic acid) to the lowest concentration that can be detected theoretically, the minimum detection ability of the reagent is verified, and the process and results are as follows.

[0157] Prepare a series of diluted samples, each concentration is repeated 6 times, calculate the CV, if the CV < 5%, it can be judged that the concentration is detectable. The experimental data are shown in Table 6 below: from Table 6 below, it can be seen that the sensitivity can reach 25 μM.

[0158] Table 6 (unit: μM)

[0159]

[0160] The beneficial effects of the embodiment mainly manifest as follows: the detection kit provided by the embodiment is designed as a liquid double reagent, so it is convenient to use and simple to operate. The reagent in the embodiment can be quickly detected on a full-automatic biochemical analyzer, and can also be used on a semi-automatic or manual instrument, so it is convenient to popularize and apply. The components participating in the coupling reaction are all added, and no additional endogenous and exogenous substance pollution is introduced. The detection kit of the embodiment has good reagent stability, strong specificity, high sensitivity, and can be used for a long time.

[0161] Embodiment 2

[0162] The embodiment provides a branched-chain amino acid detection kit and a detection method. The technical scheme comprises the following steps:

[0163] 1. Detection kit

[0164] The detection kit comprises reagent 1 and reagent 2, wherein:

[0165] The composition of reagent 1 is: 20mM pH=9.0 Tris-HCl buffer, trehalose 50g / L, EDTA 2Na 0.2g / L, Triton X-100 0.2g / L, bovine serum albumin 1g / L, ascorbate oxidase 2kU / L, leucine dehydrogenase 10kU / L, Proclin 300 1mL / L.

[0166] The composition of reagent 2 is: 50mM pH=4.0 glycine buffer, EDTA 2Na 0.5g / L, oxidized coenzyme I 25g / L, Proclin 300 1mL / L.

[0167] 2. Detection

[0168] 2.1 Detection process

[0169] The reaction temperature is set to 37°C, the reaction time is 5 minutes, the test main wavelength is 340 nm, the sample to reagent ratio is sample: reagent 1: reagent 2 = 15 μL: 90 μL: 30 μL, and the reaction is an ascending reaction. First, the sample and reagent 1 are added, and the two are automatically mixed in the automatic biochemical analyzer. The absorbance value at the main wavelength is detected and recorded. After 3 minutes, reagent 2 is added, and the two are automatically mixed in the automatic biochemical analyzer. After 5 minutes, the absorbance value at the main wavelength is recorded. According to the end-point method, the content of branched-chain amino acids in the sample is calculated by comparing the corresponding standard curve. The reaction curve of the test sample prepared by the method of this example is shown in FIG. 1. Figure 3

[0170] The detection principle is as follows:

[0171] Branched-chain amino acids (leucine, isoleucine, and valine) undergo oxidative deamination reactions under the catalysis of leucine dehydrogenase (LeuDH) to generate α-keto acid (BCKA), reduced coenzyme I, and ammonium ion. The reduced coenzyme I has a characteristic absorption peak at 340 nm, and its generation rate is linearly and positively correlated with the concentration of BCAA. The reaction equation is as follows:

[0172]

[0173] 2.2 Long-term stability verification

[0174] The kit of this example has good stability. The long-term stability experiment of the liquid reagent shows that it can be stored for 1 year at 2-8°C. The verification process and results are as follows.

[0175] (1) Sample preparation

[0176] Detection reagent: branched-chain amino acid detection reagent 1 and reagent 2 under item 1.

[0177] Calibrator: The concentration gradient of L-leucine is 0 μM, 125 μM, 250 μM, 500 μM, 1000 μM, and 2000 μM, respectively.

[0178] (2) Storage condition: After sealing, the detection reagent and the calibrator are stored in a constant temperature environment at 2-8°C to simulate the actual product storage condition.

[0179] (3) Detection time node: 0th month (initial value), 1st month, 3rd month, 5th month, 7th month, 9th month, and 12th month.

[0180] ​(4) Detection parameters: The absorbance value (OD value) of each calibration point was measured using the full-automatic biochemical analyzer IBC 900, and each concentration point was repeated 3 times, and the average value was recorded, as shown in Table 7 below.

[0181] Table 7

[0182]

[0183] (5) Stability determination standard

[0184] Taking the absorbance value measured at the 0th month (initial OD value) as the reference, the relative deviation (%) of the data at each subsequent time point (current OD value) and the initial OD value was calculated, and the formula was:

[0185]

[0186] Stability requirement: The deviation of all calibration points at each time point should be ≤10%.

[0187] The relative deviation analysis is shown in Table 8 below.

[0188] Table 8, Relative Deviation Analysis

[0189]

[0190] (6) Conclusion

[0191] Under the storage condition of 2-8°C, the branched-chain amino acid detection reagent and its calibrators showed excellent stability within 12 months, and the absorbance deviation of all calibrators (0-2000 μM) was ≤7.19%, which was significantly lower than the preset threshold of 10%. The highest deviation value appeared at the 3rd month of the 0 μM calibrator (7.19%), which still met the stability standard.

[0192] 2.3 Stability verification after opening (i.e. opening the cap)

[0193] After the reagents in the detection kit were calibrated, they were placed in the reagent compartment, and the fixed quality control was regularly detected to verify the stability performance of the reagents during use. The verification process and verification results are as follows.

[0194] (1) After opening the reagent, it was placed in the reagent compartment of the IBC 900 biochemical analyzer to simulate the actual use scene in the hospital, and the following conditions were maintained: the temperature of the reagent compartment was controlled at 2-8°C, and the humidity was controlled at 50±10% (to avoid the performance fluctuation caused by reagent moisture absorption or volatilization).

[0195] (2) Biochemical quality control (L-leucine) is a substance used to monitor the quality of the detection process, and its matrix and concentration level is close to that of clinical samples, which is periodically detected to evaluate the stability and reliability of the analysis system (instrument, reagent, operation). The present application provides branched chain amino acid high value (L-leucine concentration is 300.0 μM) and low value (L-leucine concentration is 150.0 μM) quality control for testing the bottle opening stability of reagents.

[0196] (3) Test procedure

[0197] Initial calibration: calibrate the reagent on the day of opening (day 1) and determine the baseline value of the quality control;

[0198] Daily detection: continuously measure the concentration of the quality control and calculate the deviation from the results of day 1;

[0199] Stability criteria: absolute value of deviation ≤10% is determined as stable.

[0200] The opening stability results of this example are shown in the following Table 9:

[0201] Table 9

[0202]

[0203] (4) Conclusion

[0204] The reagent is placed in the reagent compartment of the biochemical instrument after opening, and the performance is stable under the condition of 2-8℃ and humidity of 50±10%, meeting the clinical detection requirements.

[0205] 2.4 Correlation verification

[0206] The detection method of this example tests samples, and the correlation with mass spectrometry is good, and the results are shown in the following Table 10 and Figure 4 .

[0207] Table 10 (unit: μM)

[0208]

[0209] 50 clinical serum samples were tested using the detection kit and detection method of this example, and the samples have mass spectrometry results in the hospital. The correlation R 2 =0.979 between the detection method of this example and mass spectrometry is good, as shown in the following Table 10 and Figure 4 , which shows the concentration of total branched chain amino acids in the sample. Among them, the mass spectrometry method is the UHPLC-MS / MS method mentioned in Example One of the document with application number CN202410918742.6.

[0210] 2.5 Specificity verification

[0211] (1) Interfering substances L-norvaline and L-norleucine.

[0212] (2) Verification method

[0213] Prepare interfering substance solutions (L-norvaline, L-norleucine) with the same concentration (1000 μM) as the target substances (L-leucine, L-isoleucine, L-valine). Test the solution concentration using an IBC900 biochemical analyzer, calculate the recovery rate and interference rate, and use the following formulas: interference rate = test concentration / theoretical concentration * 100%; recovery rate = test concentration / theoretical concentration * 100%.

[0214] The data are shown in Table 11 below:

[0215] Table 11 (unit: μM)

[0216]

[0217] The average measured concentration of L-leucine, L-isoleucine and L-valine (target substances) was 1022.44 μM, 1047.90 μM and 968.00 (theoretical concentration 1000 μM), and the recovery rates were 101.35%, 990.5% and 97.32%, respectively, indicating that the method has high detection accuracy. L-norvaline: average measured concentration 48.96 μM, interference rate 4.90%; L-norleucine: average measured concentration 37.63 μM, interference rate 3.76%.

[0218] (3) Conclusion

[0219] The interference rates of the two interfering substances were both below the clinical detection threshold of 5%, proving that the method has excellent anti-interference ability for L-norvaline and L-norleucine.

[0220] 2.6 Sensitivity experiment

[0221] By diluting the target substance (L-leucine) to the lowest concentration that can be detected theoretically, the minimum detection ability of the reagent is verified.

[0222] Prepare a series of dilution samples, each concentration is repeated 6 times, calculate the CV, if the CV < 5%, it can be judged that the concentration is detectable. The experimental data are shown in Table 12 below: from the data in Table 12 below, it can be seen that the sensitivity can reach 31.25 μM.

[0223] Table 12 (unit: μM)

[0224]

[0225] The beneficial effects of the embodiment mainly manifest in that the reagent prepared by the method is liquid double reagent, so it is convenient to use and simple to operate. The reagent can be quickly detected on a full-automatic biochemical analyzer, and can also be used on a semi-automatic or manual instrument, so it is convenient to popularize and apply. The components participating in the coupling reaction are all added externally, and no additional endogenous and exogenous substance pollution is introduced. The detection method has good stability, strong specificity, high sensitivity, and can be used for a long time.

[0226] Example 3

[0227] The embodiment provides a branched-chain alpha-keto acid detection kit and a detection method. Compared with example 1, the difference of the embodiment is only that the pH of the Tris HCl buffer of reagent 1 is 8.5, and the rest is the same as example 1.

[0228] Detection and verification are carried out according to example 1: the 200 μM alpha-ketoisocaproic acid reaction curve is as shown in the following figure. Figure 5

[0229] The long-term stability data are shown in the following table 13.

[0230] Table 13

[0231]

[0232] The relative deviation analysis is shown in the following table 14.

[0233] Table 14, relative deviation analysis

[0234]

[0235] The post-opening stability verification result is as shown in the following figure. Figure 6

[0236] In the correlation verification, the sample is tested by the detection method of the embodiment, and compared with the mass spectrometry method. The sample correlation is good, and the sample comparison data R 2 = 0.996, as shown in the following figure. Figure 7

[0237] The specificity verification result is shown in the following table 15.

[0238] Table 15

[0239]

[0240] The sensitivity experiment result is shown in the following table 16.

[0241] Table 16 (unit: μM)

[0242]

[0243] ​​​From the results, it can be seen that the detection kit of the embodiment has a sensitivity of 25 μM.

[0244] Example 4

[0245] The embodiment provides a branched-chain alpha-keto acid detection kit and a detection method. Compared with Example 1, the only difference of the embodiment is that the pH of the Tris HCl buffer of reagent 1 is 9.5, and the rest is the same as Example 1.

[0246] Detection and verification are carried out according to Example 1. The 200 μM alpha-ketoisocaproic acid reaction curve is shown in Figure 8 .

[0247] The post-opening stability verification results are shown in Table 17.

[0248] Table 17

[0249]

[0250] In the correlation verification, the detection method of the embodiment is used to test samples, and the mass spectrometry method is used for comparison. The samples have good correlation, and the sample comparison data R 2 = 0.981, as shown in Figure 9 .

[0251] The results of the specificity verification are shown in Table 18.

[0252] Table 18 (unit: μM)

[0253]

[0254] From the data in Table 18, it can be seen that the average measured concentration of alpha-ketoisocaproic acid (target) is 244.5 μM (theoretical concentration 250 μM), and the recovery rate is 97.8%, indicating that the method has high detection accuracy. Pyruvic acid (non-branched-chain alpha-keto acid): average measured concentration 9.7 μM, interference rate 3.89%; beta-ketoisocaproic acid (abnormal metabolic keto acid): average measured concentration 12.3 μM, interference rate 4.92%. Conclusion: The interference rates of the two types of interferents are lower than the clinical detection threshold of 5%, proving that the method has excellent anti-interference ability to structural analogs and metabolic related interferents.

[0255] Example 5

[0256] The embodiment provides a branched-chain amino acid detection kit and a detection method. Compared with Example 2, the only difference of the embodiment is that the pH of the Tris HCl buffer of reagent 1 is 8.5, and the rest is the same as Example 2.

[0257] According to the content described in Example 2 under the item “2.4 Correlation verification”, the embodiment and the mass spectrometry method are compared.

[0258] Table 19 (unit: μM)

[0259]

[0260] As shown in Table 19 and Figure 10 above, the correlation R 2 = 0.996 of the present example with the mass spectrometry sample is good.

[0261] Example 6

[0262] The branched chain amino acid detection kit and detection method of the present example are the same as those of Example 2, except that the pH of the Tris-HCl buffer of reagent 1 is 9.5.

[0263] The present example was compared with the mass spectrometry method according to the description under the item "2.4 Correlation verification" of Example 2. The results are shown in Table 20 and Figure 11 below.

[0264] Table 20 (unit: μM)

[0265]

[0266] The results show that the correlation R 2 = 0.994 of the present example with the mass spectrometry sample is good.

[0267] Comparative Example 1

[0268] The branched chain α-keto acid detection kit and detection method of the present comparative example are the same as those of Example 1, except that the Tris-HCl buffer of reagent 1 is 30 mM, pH = 9.0 Tris-HCl buffer.

[0269] Detection and verification were carried out according to Example 1:

[0270] The long-term stability data are shown in Table 21. According to the data in Table 21, the absorbance deviation of all the calibration points (0-400 μM) is lower than the preset threshold of 10% under the storage condition of 2-8°C, and meets the stability standard.

[0271] Table 21

[0272]

[0273] The relative deviation analysis results are shown in Table 22:

[0274] Table 22, Relative Deviation Analysis

[0275]

[0276] In the correlation verification, the sample was tested by the detection method of the present comparative example, and compared with the mass spectrometry. The sample correlation was poor, and the sample comparison data R 2 = 0.835, as shown in Table 23. Figure 12 According to the description in the item of "2.5 Specificity verification" in Example 1, the specificity of the detection kit and the detection method of the present comparative example were verified, and the results are shown in Table 23.

[0277] Table 23 (μM)

[0278]

[0279] From the data in Table 23, it can be seen that the specificity of the detection kit and the detection method of the present comparative example was poor, the average measured concentration of a-ketoisocaproic acid (target substance) was 237.9 μM (theoretical concentration 250 μM), and the recovery rate was 95.16%, indicating that the method had high detection accuracy. Pyruvic acid (non-branched α-keto acid): the average measured concentration was 20.8 μM, and the interference rate was 8.32%; β-ketoisocaproic acid (abnormal metabolic keto acid): the average measured concentration was 47.6 μM, and the interference rate was 19.05%. Conclusion: The interference rates of the two types of interferents were higher than the clinical detection threshold of 5%, indicating that the anti-interference ability decreased.

[0280] Comparative Example 2

[0281] The present comparative example provides a branched chain amino acid detection kit and a detection method. Compared with Example 2, the difference of the present comparative example is only that the Tris-HCl buffer in reagent 1 is replaced by 20 mM PB buffer with pH = 9.0.

[0282] Detection and verification were carried out according to Example 2:

[0283] The detection results of long-term stability are shown in Table 24. According to the data in Table 24, under the storage condition of 2-8℃, the absorbance deviation of all calibration points (0-2000 μM) was lower than the preset threshold of 10%, meeting the stability standard.

[0284] Table 24

[0285]

[0286] The relative deviation analysis results are shown in Table 25:

[0287] Table 25, relative deviation analysis

[0288]

[0289] The verification results of the stability after opening the bottle are shown in Table 26, from which it can be seen that the absolute value of the deviation is ≤10%, and it is determined to be stable.

[0290] Table 26

[0291]

[0292] The results of the correlation verification are shown in Tables 27 and Figure 13

[0293] Table 27 (unit: μM)

[0294]

[0295] The results show that the correlation of the detection method of the present comparative example and the mass spectrometry is poor, and R 2 decreases to 0.904.

[0296] The specificity verification results are shown in Table 28:

[0297] Table 28 (unit: μM)

[0298]

[0299] The sensitivity detection results are shown in Table 29:

[0300] Table 29

[0301]

[0302] Each technical feature of the above-described embodiments and examples can be combined in any suitable manner. In order to make the description simple, each technical feature in the above-described embodiments and examples is not described in all possible combinations, however, as long as the combination of the technical features does not exist contradictions, it should be considered within the scope of the present disclosure.

[0303] ​The above-described embodiments only express several implementation manners of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the patent protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. In addition, it should be understood that, after reading the above teaching content of the present application, the skilled person in the art can make various modifications or modifications to the present application, and the equivalent forms obtained are also within the protection scope of the present application. It should also be understood that, on the basis of the technical solutions provided by the present application, the skilled person in the art obtains the technical solutions through logical analysis, reasoning or limited experiments, and all of them are within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be based on the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A kit characterized in that, The kit comprises reagent 1 and reagent 2; The reagent 1 comprises 20 mM Tris-HCl buffer with pH of 8.5-9.5, 50 g / L trehalose, 0.2 g / L EDTA 2Na, 0.2 g / L Triton X-100, 1 g / L bovine serum albumin, 2 kU / L ascorbate oxidase, 10 kU / L leucine dehydrogenase, 1 g / L Proclin 300, and 1 g / L reduced coenzyme I; The reagent 2 comprises 50 mM glycine buffer with pH of 4.0, 0.5 g / L EDTA 2Na, 20 g / L ammonium sulfate, and 1 g / L Proclin 300; The amino acid sequence of the leucine dehydrogenase is shown in SEQ ID NO.

1.

2. A kit characterized in that, The kit comprises reagent 1 and reagent 2; The reagent 1 comprises 20 mM Tris-HCl buffer with pH of 8.5-9.5, 50 g / L trehalose, 0.2 g / L EDTA 2Na, 0.2 g / L Triton X-100, 1 g / L bovine serum albumin, 2 kU / L ascorbate oxidase, 10 kU / L leucine dehydrogenase, and 1 mL / L Proclin 300; The reagent 2 comprises 50 mM glycine buffer with pH of 4.0, 0.5 g / L EDTA 2Na, 25 g / L oxidized coenzyme I, and 1 mL / L Proclin 300; The amino acid sequence of the leucine dehydrogenase is shown in SEQ ID NO.

1.

3. A method of detecting branched-chain alpha-keto acids, which is not directly aimed at obtaining a disease diagnostic result or a health condition, characterized by, The detection method uses the kit of claim 1 to detect the sample to be tested.

4. A method for detecting branched chain amino acids, which is not directly aimed at obtaining a disease diagnosis result or a health condition, characterized by, The detection method uses the kit of claim 2 to detect the sample to be tested.

5. The detection method according to claim 3 or 4, characterized in that, The detection method meets one or more of the following conditions: 1) the detection method comprises ultraviolet-visible spectrophotometry; and, 2) during the detection process, the volume ratio of the sample to be tested, the reagent 1 and the reagent 2 is (10-25):(85-95):(25-35).

6. The detection method according to claim 5, characterized in that, The detection wavelength used by the detection method is 340 nm.

7. The detection method according to claim 3 or 4, characterized by, During the detection process, the reaction temperature is 37℃.

8. The detection method according to claim 3 or 4, characterized by, During the detection process, the reaction time is 5 minutes.

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