Kit and method of making same

Abstract

The present application relates to the field of biochemical detection, in particular to a kit and a preparation method thereof. The present application provides a lactate dehydrogenase method for determining a pyruvic acid detection kit. The kit comprises reagent R1 and reagent R2; the reagent R1 is composed of buffer, stabilizer, surfactant, preservative, nicotinamide adenine dinucleotide (NADH); the reagent R2 is composed of buffer, preservative, lactate dehydrogenase (LDH); the test shows that the kit has high accuracy of detection result, strong anti-interference ability, wide linear detection range, good stability, long shelf life, and is easy to popularize in clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemical detection, in particular to a kit and a preparation method thereof. BACKGROUND

[0002] Pyruvic acid is an important intermediate product of glycolysis and anabolism in vivo. Pyruvic acid is a product of glycolysis pathway, and is oxidized to CO2 and H2O through the tricarboxylic acid cycle, so that the lactic acid / pyruvic acid ratio in blood is maintained at about 9. When the body is in a hypoxic metabolism condition, pyruvic acid is reduced to lactic acid, and the lactic acid / pyruvic acid ratio increases. The more serious the hypoxia, the more obvious the increase in the ratio. The severity of circulatory failure can be inferred according to the ratio, and severe cases can lead to lactic acid poisoning. Mild activity can cause both lactic acid and pyruvic acid to increase, but the ratio does not change. The determination of blood pyruvic acid is mainly used for the diagnosis of vitamin B1 deficiency. The pyrophosphate ester of vitamin B1 is a decarboxylation coenzyme when pyruvic acid is further oxidized and decomposed into acetyl coenzyme A in cells. When vitamin B1 is deficient, the oxidation of pyruvic acid in the body is impaired, resulting in an increase in the content of pyruvic acid; an increase in blood pyruvic acid is also seen in diabetes, heart failure, diarrhea, severe liver damage, acute infection, chronic alcoholism, chronic pulmonary heart disease and ketoacidosis, etc., so the determination of the content of pyruvic acid has very important clinical significance.

[0003] At present, the main methods for detecting pyruvic acid are: dinitrophenylhydrazine method, enzyme-linked immunoassay, liquid phase analysis method, chemiluminescence method and lactate dehydrogenase method. The dinitrophenylhydrazine colorimetric method is based on the reaction of pyruvic acid with 2,4-dinitrophenylhydrazine to form colored benzhydrazine, but this method is interfered by other alpha-keto acids, has poor specificity, and is complicated to operate. The enzyme-linked immunoassay has high sensitivity and strong specificity, but has many interference factors and poor repeatability. The liquid phase and chemiluminescence have good accuracy and sensitivity, but have high cost, and the liquid phase analysis method requires sample pretreatment and takes a long time to operate.

[0004] The lactate dehydrogenase method has the advantages of simple operation, rapidness, sensitivity, rapid high-throughput determination by automatic biochemical analyzer, and low cost. Therefore, the development of a pyruvic acid detection reagent suitable for clinical application in large, medium and small hospitals and grass-roots units will contribute to disease diagnosis, treatment and prognosis. SUMMARY

[0005] Therefore, the present application provides a kit and a preparation method thereof. Compared with conventional kits, the kit has better anti-interference and linear range, high stability and precision level, and is conducive to the popularization and application of the reagent in clinical practice.

[0006] In order to achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0007] The application provides the following any one in improving the anti-interference ability and / or accuracy of a pyruvic acid detection reagent and / or kit:

[0008] (I) increase of pH value; or

[0009] (II) change of stabilizer and surfactant concentration;

[0010] The anti-interference ability includes anti-hemoglobin interference ability and / or anti-fat milk interference ability;

[0011] The pH value is the pH value of a reagent containing the stabilizer in the pyruvic acid detection reagent and / or kit; and / or

[0012] The changed concentration of the stabilizer is 20-40 g / L; and / or

[0013] The changed concentration of the surfactant is 20-40 g / L; and / or

[0014] The stabilizer includes NaCl; and / or

[0015] The surfactant includes one or a combination of two or more of alkyl polyethylene hydroxy ethanol, polydimethylsiloxane or fatty alcohol ethoxylate; and / or

[0016] The alkyl polyethylene hydroxy ethanol includes 15-S-9 and / or 15-S-30; and / or

[0017] The polydimethylsiloxane includes methyl ethyl oxide SILWET L7600; and / or

[0018] The fatty alcohol ethoxylate includes X-080.

[0019] The application also provides a reagent combination including reagent 1 and reagent 2.

[0020] The reagent 1 includes, in terms of concentration:

[0021]

[0022] The reagent 2 includes, in terms of concentration:

[0023] The buffer 2 is 50-100 mM;

[0024] The preservative 2 is 0.5-1.0 g / L;

[0025] The lactate dehydrogenase is 250-400 kU / L;

[0026] The pH value of the reagent 1 is 9.0-10.0;

[0027] the pH of the reagent 2 is 7.0-8.0;

[0028] the buffer 1 and the buffer 2 can be the same or different;

[0029] the preservative 1 and the preservative 2 can be the same or different.

[0030] In some embodiments of the present application, the buffer 1 or the buffer 2 in the above reagent combination is independently selected from one or a combination of more than one of CAPSO buffer, AMP buffer, CHES buffer, triethanolamine buffer, MOPS buffer, HEPES buffer, TAPSO buffer, MOBS buffer, or TES buffer; and / or

[0031] the pH of the buffer 1 is 9.0-10.0; and / or

[0032] the pH of the buffer 2 is 7.0-8.0; and / or

[0033] the stabilizer comprises NaCl; and / or

[0034] the surfactant comprises one or a combination of more than one of alkoxypolyethylene hydroxy ethanol, polydimethylsiloxane, or fatty alcohol ethoxylate; and / or

[0035] the preservative 1 or the preservative 2 is independently selected from PC300 and / or NaN3; and / or

[0036] the alkoxypolyethylene hydroxy ethanol comprises 15-S-9 and / or 15-S-30; and / or

[0037] the polydimethylsiloxane comprises methyl ethyl oxide SILWET L7600; and / or

[0038] the fatty alcohol ethoxylate comprises X-080.

[0039] In some embodiments of the present application, the buffer 1 in the above reagent combination is CAPSO buffer; and / or

[0040] the buffer 2 is triethanolamine; and / or

[0041] the stabilizer is NaCl; and / or

[0042] the surfactant is X-080; and / or

[0043] the preservative 1 is PC300; and / or

[0044] The preservative 2 is NaN3.

[0045] In some embodiments of the present application, the reagent combination further comprises a calibrator.

[0046] The calibrator is obtained by mixing sodium pyruvate and a buffer.

[0047] The concentration of the calibrator comprises 200 μmol / L.

[0048] The present application further provides the use of the reagent combination in the preparation of a pyruvate detection kit.

[0049] The present application further provides a kit comprising the reagent combination and acceptable adjuvants or auxiliaries.

[0050] In some embodiments of the present application, the kit comprises reagent 1 and reagent 2.

[0051] The reagent 1 comprises, in terms of concentration:

[0052]

[0053] The reagent 2 comprises, in terms of concentration:

[0054]

[0055] The pH value of the reagent 1 is 9.0-10.0.

[0056] The pH value of the reagent 2 is 7.0-8.0.

[0057] In some embodiments of the present application, the pH value of the CAPSO in the kit comprises 9.5; and / or

[0058] The pH value of the triethanolamine comprises 7.5.

[0059] In some embodiments of the present application, the kit further comprises a calibrator.

[0060] The calibrator is obtained by mixing sodium pyruvate and a buffer.

[0061] The concentration of the calibrator comprises 200 μmol / L.

[0062] The kit of the present application has the following effects:

[0063] The present application adopts lactate dehydrogenase method, optimizes the reaction system, improves the accuracy and stability of the reagent measurement value, and expands the linear detection range.

[0064] The correlation experiment results show that the optimized NaCl and surfactant The X-080 content, and the combination of the two can improve the accuracy of the reagent measurement value.

[0065] The precision test results show that the kit has good precision.

[0066] The anti-interference test results show that the content of the preferred stabilizer NaCl and surfactant can significantly improve the anti-fat emulsion ability of the reagent; NaCl can improve the anti-hemoglobin ability of the reagent, and the anti-hemoglobin ability is enhanced with the increase of pH; and the coordination of NaCl and pH can significantly improve the anti-hemoglobin ability of the reagent.

[0067] The stability test results show that the relative deviation is small before and after 18 months of storage at 2-8 DEG C. BRIEF DESCRIPTION OF DRAWINGS

[0068] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below.

[0069] Figure 1 The correlation relationship diagram of the detection results of the detection kit and the control kit of the present application is shown;

[0070] Figure 2 The linear relationship diagram of the pyruvic acid concentration detection value and the theoretical value of the detection kit of the present application is shown. DETAILED DESCRIPTION

[0071] The present application discloses a kit and a preparation method thereof, and those skilled in the art can refer to the content of the present application, and appropriately improve the process parameters. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are regarded as included in the present application. The method and application of the present application have been described by the preferred embodiments, and the related personnel can obviously modify or appropriately change and combine the methods and applications described in the present application without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0072] The lactate dehydrogenase method for determining pyruvic acid provided by the present application comprises:

[0073] 1) Calibration: the sodium pyruvate pure product is added to the buffer to prepare, and the single-point calibration concentration is 200 μmol / L;

[0074] 2) R1 reagent components include: buffer A 50-100 mM; stabilizer 20-40 g / L; surfactant 20-40 g / L; preservative 0.1-0.2 g / L; NADH 0.3-0.5 mmol / L;

[0075] 3) R2 reagent components include: buffer B 50-100 mM; preservative 0.5-1.0 g / L; LDH 250-400 kU / L;

[0076] The buffer A is any one of CAPSO buffer, AMP buffer, CHES buffer, triethanolamine buffer, and the buffer B is any one of triethanolamine, MOPS, HEPES, TAPSO, MOBS or TES buffer;

[0077] The pH of the buffer A is 9.0-10.0, and the pH of the buffer B is 7.0-8.0;

[0078] The stabilizer is NaCl;

[0079] The surfactant is alkoxypolyethylenehydroxy ethanol 15-S-9, alkoxypolyethylenehydroxy ethanol 15-S-30, polydimethylsiloxane methyl ethyl oxide SILWET L7600 or fatty alcohol ethoxylate one or more of X-080.

[0080] The innovation of the present application is as follows:

[0081] The preferred NaCl and surfactant content, and the synergistic effect of NaCl and surfactant, improve the accuracy of reagent measurement value.

[0082] The preferred NaCl and surfactant content, and the synergistic effect of NaCl and surfactant, improve the accuracy of reagent measurement value.

[0083] The preferred R1 reagent pH, NaCl synergistic effect, significantly improves the anti-hemoglobin capacity of the reagent.

[0084] Unless otherwise specified, the raw materials, reagents, consumables and instruments used in the present application are ordinary commercially available products, which can be purchased from the market.

[0085] The present application will be further described below in conjunction with examples:

[0086] Example 1

[0087] 1) The component content of reagent R1:

[0088]

[0089] 2) The component content of reagent R2:

[0090]

[0091] The preparation of the reagent R1 and reagent R2 is as follows:

[0092] Reagent R1 preparation:

[0093] According to the formula, 23.73 g of CAPSO, 30 g of NaCl, 30 g of X-080, 0.1 g of PC300 were weighed in a clean container, stirred with deionized water for 30 min, fully dissolved, adjusted to pH 9.5; 0.199 g of NADH was added, fully stirred to uniform; deionized water was added to 1 L, filtered to obtain R1.

[0094] Reagent R2 preparation:

[0095] According to the formula, 14.92 g of triethanolamine, 0.5 g of NaN3 were weighed in a clean container, stirred with deionized water for 30 min, fully dissolved, adjusted to pH 7.0; 0.848 g of LDH was added, fully stirred to uniform; deionized water was added to 1 L, filtered to obtain R2.

[0096] The pyruvic acid detection kit described in this example, the determination method is to use Toshiba 120 automatic analyzer, the operation is as follows: add 16 μL of deionized water, sample or calibrator, then add 160 μL of R1 reagent, mix well, 37°C constant temperature 5 min, read the absorbance A1 relative to the blank, then add 40 μL of reagent R2, mix well, 37°C constant temperature 5 min, read the absorbance A2, and calculate ΔA = A2-A1.

[0097] Example 2

[0098] 1) The component content of reagent R1:

[0099]

[0100] 2) The component content of reagent R2:

[0101]

[0102] Reagent R1 preparation:

[0103] According to the formula, 23.73 g of CAPSO, 30 g of NaCl, 30 g of X-080, 0.2 g of PC300 were weighed in a clean container, stirred with deionized water for 30 min, fully dissolved, adjusted to pH 9.5; 0.265 g of NADH was added, fully stirred to uniform; deionized water was added to 1 L, filtered to obtain R1.

[0104] Reagent R2 preparation:

[0105] Weigh 14.92g of triethanolamine and 1.0g of NaN3 into a clean container according to the formula, add deionized water and stir for 30 minutes to fully dissolve them, and adjust the pH to 7.5; add 0.989g of LDH and stir thoroughly until homogeneous; add deionized water to make up to 1L, and filter to obtain R2.

[0106] Example 3

[0107] 1) Component content of reagent R1:

[0108]

[0109]

[0110] 2) Component content of reagent R2:

[0111]

[0112] Preparation of reagent R1:

[0113] Weigh out 11.865g of CAPSO, 20g of NaCl, and 40g of [unspecified ingredient] according to the formula. In a clean container, add X-080 and 0.1g of PC300, add deionized water and stir for 30 minutes to fully dissolve them, and adjust the pH to 9.0; add 0.331g of NADH and stir thoroughly until homogeneous; add deionized water to make up to 1L, and filter to obtain R1.

[0114] Preparation of reagent R2:

[0115] Weigh 7.46g of triethanolamine and 0.5g of NaN3 into a clean container according to the formula, add deionized water and stir for 30 minutes to fully dissolve them, and adjust the pH to 8.0; add 1.131g of LDH and stir thoroughly until homogeneous; add deionized water to make up to 1L, and filter to obtain R2.

[0116] Example 4

[0117] 1) Component content of reagent R1:

[0118]

[0119] 2) Component content of reagent R2:

[0120]

[0121] Preparation of reagent R1:

[0122] Weigh out 23.73g of CAPSO, 40g of NaCl, and 20g of [unspecified ingredient] according to the formula. X-080, 0.1 g of PC300 in a clean container, stirring with deionized water for 30 min, make it fully dissolved, adjust the pH to 10.0; add 0.232 g of NADH, fully stirred to uniform; add deionized water to 1 L, filter to get R1.

[0123] Reagent R2 preparation:

[0124] According to the formula, 14.92 g of triethanolamine, 0.5 g of NaN3 in a clean container, stirring with deionized water for 30 min, make it fully dissolved, adjust the pH to 7.0; add 0.848 g of LDH, fully stirred to uniform; add deionized water to 1 L, filter to get R2.

[0125] Example 5

[0126] 1) Component content of reagent R1:

[0127]

[0128] 2) Component content of reagent R2:

[0129]

[0130] The preparation of the reagent R1, reagent R2 is as follows:

[0131] Reagent R1 preparation:

[0132] According to the formula, 17.798 g of CAPSO, 30 g of NaCl, 30 g of X-080, 0.1 g of PC300 in a clean container, stirring with deionized water for 30 min, make it fully dissolved, adjust the pH to 9.5; add 0.199 g of NADH, fully stirred to uniform; add deionized water to 1 L, filter to get R1.

[0133] Reagent R2 preparation:

[0134] According to the formula, 11.19 g of triethanolamine, 1.0 g of NaN3 in a clean container, stirring with deionized water for 30 min, make it fully dissolved, adjust the pH to 7.5; add 0.848 g of LDH, fully stirred to uniform; add deionized water to 1 L, filter to get R2.

[0135] Comparative Example 1

[0136] A test kit for determining pyruvate using the lactate dehydrogenase method includes reagents R1 and R2, consistent with Example 1, except that: reagent R1 includes the following components: 100mM buffer solution at pH 9.5; 30g / L NaCl stabilizer; 0.1g / L preservative; and 0.3mmol / L NADH.

[0137] The preparation method of the R1 reagent includes:

[0138] Weigh 23.73g of CAPSO, 30g of NaCl, and 0.1g of PC300 into a clean container according to the formula. Add deionized water and stir for 30 minutes to fully dissolve the substances. Adjust the pH to 9.5. Add 0.199g of NADH and stir thoroughly until homogeneous. Add deionized water to a final volume of 1L and filter to obtain R1.

[0139] Comparative Example 2

[0140] A detection kit for determining pyruvate using the lactate dehydrogenase method includes reagent R1 and reagent R2, consistent with Example 1, except that: reagent R1 includes the following components: 100mM buffer solution at pH 9.5; 30g / L surfactant; 0.1g / L preservative; 0.3mmol / L NADH;

[0141] The preparation method of the R1 reagent includes:

[0142] Weigh out 23.73g of CAPSO and 30g of [unspecified ingredient] according to the formula. In a clean container, add X-080 and 0.1g of PC300, add deionized water and stir for 30 minutes to fully dissolve them, and adjust the pH to 9.5; add 0.199g of NADH and stir thoroughly until homogeneous; add deionized water to make up to 1L, and filter to obtain R1.

[0143] Comparative Example 3

[0144] A detection kit for determining pyruvate using the lactate dehydrogenase method includes reagent R1 and reagent R2, consistent with Example 1, except that: reagent R1 includes the following components: 100mM buffer solution at pH 9.5; 30g / L NaCl stabilizer; 10g / L surfactant; 0.1g / L preservative; and 0.3mmol / L NADH.

[0145] The preparation method of the R1 reagent includes:

[0146] Weigh out 23.73g of CAPSO, 30g of NaCl, and 10g of [unspecified ingredient] according to the formula. X-080, 0.1 g of PC300 in a clean container, stirring with deionized water for 30 min, make it fully dissolved, adjust the pH to 9.5; add 0.199 g of NADH, fully stir to uniform; add deionized water to 1 L, filter to get R1.

[0147] Comparative Example 4

[0148] A lactate dehydrogenase method for determining pyruvic acid test kit, including R1 reagent and R2 reagent, consistent with example 1, the difference is that: the R1 reagent includes the following components: buffer 100 mM of pH 9.5; stabilizer NaCl 10 g / L; surfactant 30 g / L; preservative 0.1 g / L; NADH 0.3 mmol / L;

[0149] The preparation method of the R1 reagent includes:

[0150] According to the formula, 23.73 g of CAPSO, 10 g of NaCl, 30 g of X-080, 0.1 g of PC300 in a clean container, stirring with deionized water for 30 min, make it fully dissolved, adjust the pH to 9.5; add 0.199 g of NADH, fully stir to uniform; add deionized water to 1 L, filter to get R1.

[0151] Comparative Example 5

[0152] A lactate dehydrogenase method for determining pyruvic acid test kit, including R1 reagent and R2 reagent, consistent with example 1, the difference is that: the R1 reagent includes the following components: buffer 100 mM of pH 8.5; stabilizer NaCl 30 g / L; preservative 0.1 g / L; NADH 0.3 mmol / L;

[0153] The preparation method of the R1 reagent includes:

[0154] According to the formula, 23.73 g of CAPSO, 30 g of NaCl, 0.1 g of PC300 in a clean container, stirring with deionized water for 30 min, make it fully dissolved, adjust the pH to 8.5; add 0.199 g of NADH, fully stir to uniform; add deionized water to 1 L, filter to get R1.

[0155] Comparative Example 6

[0156] A lactate dehydrogenase method for determining pyruvic acid detection kit, including R1 reagent and R2 reagent, consistent with example 1, the difference lies in: the R1 reagent includes the following components: buffer 100mM of pH 8.5;Stabilizer NaCl 30g / L;Surfactant 30g / L;Preservative 0.1g / L;NADH 0.3mmol / L;

[0157] The preparation method of the R1 reagent comprises:

[0158] According to the formula, 23.73g of CAPSO, 30g of NaCl, 30g of X-080, 0.1g of PC300 are weighed in a clean container, stirred with deionized water for 30min, fully dissolved, adjust pH to 8.5;Add 0.199g of NADH, fully stir until uniform;Add deionized water to 1L, filter to get R1.

[0159] Comparative example 7

[0160] A lactate dehydrogenase method for determining pyruvic acid detection kit, including R1 reagent and R2 reagent, consistent with example 1, the difference lies in: the R1 reagent includes the following components: buffer 100mM of pH 7.5;Stabilizer NaCl 30g / L;Surfactant 30g / L;Preservative 0.1g / L;NADH 0.3mmol / L;

[0161] The preparation method of the R1 reagent comprises:

[0162] According to the formula, 23.73g of CAPSO, 30g of NaCl, 30g of X-080, 0.1g of PC300 are weighed in a clean container, stirred with deionized water for 30min, fully dissolved, adjust pH to 7.5;Add 0.199g of NADH, fully stir until uniform;Add deionized water to 1L, filter to get R1.

[0163] Comparative example 8

[0164] Patent CN106290212A A kind of pyruvic acid detection reagent with high sensitivity.

[0165] The components and corresponding contents included in the reagent R1 are: Tris 10mmol / L, NADPH 2mmol / L, chitosan 1g / L, Surfynol485 2mL / L, Zonyl FSN100 0.5mL / L, lithium chloride 0.1g / L, dihydroxyethyl glycine (DEG) 1mol / L, sodium azide 0.1g / L, pH 9.5.

[0166] The reagent R2 comprises the following components and corresponding contents: phosphate buffer 100 mmol / L, trehalose 5 g / L, lactate dehydrogenase 2 kU / L, sodium azide 0.1 g / L, pH 6.5.

[0167] Comparative Example 9

[0168] Patent CN109212176A Pyruvic acid determination kit and preparation method and application thereof.

[0169] The reagent R1 comprises the following components and corresponding contents: Tris 80 mmol / L, NADH 0.35 mmol / L, EDTA-Na2 10 mmol / L, triton-100 1 g / L, NaN3 1 g / L, pH 8.0.

[0170] The reagent R2 comprises the following components and corresponding contents: Tris 80 mmol / L, lactate dehydrogenase (LDH) 300 U / L, polyethylene glycol 6000 5 g / L, BSA 5 g / L, glucomannan 10 g / L, triton-100 1 g / L, NaN3 1 g / L, pH 6.0.

[0171] Comparative Example 10

[0172] Patent CN109212176A Pyruvic acid determination kit and preparation method and application thereof.

[0173] The reagent R1 comprises the following components and corresponding contents: Tris 80 mmol / L, NADH 0.35 mmol / L, EDTA-Na2 10 mmol / L, Tween 201 g / L, NaN3 1 g / L, pH 8.0.

[0174] The reagent R2 comprises the following components and corresponding contents: Tris 80 mmol / L, lactate dehydrogenase (LDH) 300 U / L, polyethylene glycol 6000 5 g / L, BSA 5 g / L, glucomannan 10 g / L, Tween 20 1 g / L, gentamicin sulfate 1 g / L, pH 6.0.

[0175] Comparative Example 11

[0176] Patent CN109212176A Pyruvic acid determination kit and preparation method and application thereof.

[0177] The reagent R1 comprises the following components and corresponding contents: Tris 80 mmol / L, reduced coenzyme I (NADH) 0.35 mmol / L, EDTA-Na2 10 mmol / L, Brij35 1 g / L, NaN3 1 g / L, pH 8.0.

[0178] The reagent R2 comprises the following components and corresponding contents: Tris 80 mmol / L, lactate dehydrogenase (LDH) 300 U / L, polyethylene glycol 6000 5 g / L, BSA 5 g / L, glucomannan 10 g / L, Tween 20 1 g / L, NaN3 1 g / L, pH 6.0.

[0179] Effect example

[0180] The performance evaluation results of the reagent kits of Examples 1-5 are basically consistent. The reagent kits prepared in Examples 1-5 are taken as examples to verify the correlation, linear range, precision, anti-interference and stability and other related performances.

[0181] (1) Standard curve preparation

[0182] The reagent of the above implementation method is tested by Toshiba 120FR full-automatic biochemical instrument, the test wavelength is 340 nm / 404 nm, 16 μL of sample or calibrant is taken, 160 μL of reagent R1 is added, 37℃ constant temperature for 5 min, and the absorbance A1 is read relative to the blank; then 40 μL of reagent R2 is added, the absorbance A2 is read after 37℃ incubation for 5 min, and then the reaction absorbance ΔA = A2-A1; the standard sample is used for multi-point calibration, and the spline function is used for calculation to obtain the calibration curve, and the concentration value of pyruvic acid in the sample can be found from the working curve.

[0183] (2) Correlation experiment

[0184] A recognized accuracy of acetone kit on the market (Beijing nine strong biological technology Co. Ltd.) as a control group, the kit of the example as experimental group were compared, 40 samples were detected, and the results were as shown in Table 1. The test results of the control kit on the market were taken as the horizontal coordinate independent variable (control group), and the test results of the kit of the application were taken as the vertical coordinate dependent variable, a linear regression curve was drawn, the regression equation of Example 1 was Y = 0.9943X + 0.3725, the linear correlation coefficient R = 0.9977; the regression equation of Example 2 was Y = 1.0079X - 1.037, the linear correlation coefficient R = 0.9958; the regression equation of Example 3 was Y = 0.998X - 0.2216, the linear correlation coefficient R = 0.9957; the regression equation of Example 4 was Y = 0.9957X - 0.5524, the linear correlation coefficient R = 0.996; the regression equation of Example 5 was Y = 1.0001X - 1.2058, the linear correlation coefficient R = 0.9952; the linear relationship was good, and the test results could be effectively used as clinical examination. The correlation curve of Example 1 was shown in Figure 1 The experimental results of Comparative Examples 1-4 were shown in Table 2; the regression equation of Comparative Example 1 was Y = 0.9385X - 11.617, the linear correlation coefficient R = 0.978; the regression equation of Comparative Example 2 was Y = 0.9719X - 6.7645, the linear correlation coefficient R = 0.9765; the regression equation of Comparative Example 3 was Y = 0.9446X - 4.5534, the linear correlation coefficient R = 0.9761; the regression equation of Comparative Example 4 was Y = 0.9789X - 2.2322, the linear correlation coefficient R = 0.9755; the results of Tables 1-2 showed that the optimized NaCl and surfactant X-080 content, and the two could improve the accuracy of the reagent measurement value.

[0185] Table 1: Detection results of correlation experiments of Examples 1-5 (unit: μmol / L)

[0186]

[0187]

[0188] Table 2: Detection results of correlation experiments of Comparative Examples 1-4 (unit: μmol / L)

[0189]

[0190]

[0191] (3) Linear experiment

[0192] The sodium pyruvate high value sample 2160 μmol / L is diluted to prepare 6 different gradient concentration samples, which are 2160 μmol / L, 1620 μmol / L, 1080 μmol / L, 540 μmol / L, 270 μmol / L, 135 μmol / L and 0 μmol / L in turn, and each sample is measured twice to take the average. The linear determination results are shown in Table 3. The linear regression equation is obtained by taking the theoretical concentration as the independent variable X and the actual test value as the dependent variable Y, and the linear regression correlation coefficient r is calculated. The results show that the linear regression equation of Example 1 is Y = 1.0152X-2.3516, the correlation coefficient r is 0.9999; the linear regression equation of Example 2 is Y = 0.996X+5.0023, the correlation coefficient r is 0.9998; the linear regression equation of Example 3 is Y = 1.0022X-0.1294, the correlation coefficient r is 0.9997; the linear regression equation of Example 4 is Y = 1.0062X-1.705, the correlation coefficient r is 0.9998; the linear regression equation of Example 5 is Y = 1.0061X-0.5938, the correlation coefficient r is 0.9998; which shows that the correlation of the application is good in the linear range of 0-2160 μmol / L. The linear range curve of Example 1 is as follows: Figure 2 .

[0193] Table 3 Linear range analysis results of Examples 1-5

[0194]

[0195]

[0196] (4) Precision detection

[0197] Two serum samples with high and low pyruvic acid (PYR) concentrations are respectively continuously tested for 20 times, and the coefficient of variation is calculated. The precision results of the examples are shown in Table 4. The reagent kit has good precision.

[0198] Table 4 Precision detection results of Examples 1-5

[0199]

[0200] (5) Anti-interference detection

[0201] The clinical normal patient serum was divided into two parts, one part was added with the highest concentration of interference substance, and the other part was added with the same amount of solvent. The samples with and without the addition of interference substance were subjected to 3 gradient equal difference dilutions, each sample was detected three times, the detection order was reversed, and the deviation of the measured value was calculated. The anti-fat emulsion interference ability of the reagent of the example was evaluated, and the results were shown in Table 5; the anti-fat emulsion interference ability of the reagents of Comparative Examples 1-4, and the anti-fat emulsion interference ability of the reagents of Comparative Examples 8-11 were evaluated, and the results were shown in Tables 6 and 7. The anti-hemoglobin interference ability of the reagents of Example 1, Comparative Examples 1-2 and 5-7 was evaluated, and the results were shown in Table 8. The results of Tables 5-6 showed that: NaCl and surfactant The use of the complex of X-080 significantly improved the anti-fat emulsion ability of the reagent; the use of NaCl or X-080 alone had poor anti-interference ability; and when the content was insufficient, the anti-interference ability was also poor. The results of Table 7 showed that: the anti-interference ability of the reagent kits of Comparative Examples 8-11 was poor, and the anti-fat emulsion interference ability of the reagent kit was significantly better than that of Comparative Examples 8-11. The results of Table 8 showed that: NaCl could improve the anti-hemoglobin ability of the reagent, and the anti-hemoglobin ability was enhanced with the increase of pH; and the coordination of NaCl and pH significantly improved the anti-hemoglobin ability of the reagent.

[0202] Table 5 Anti-fat emulsion interference detection results of reagents of Examples 1-5

[0203]

[0204] Table 6 Anti-fat emulsion interference detection results of reagents of Comparative Examples 1-4

[0205]

[0206] Table 7 Anti-fat emulsion interference detection results of reagents of Comparative Examples 8-11

[0207]

[0208] Table 8 Anti-hemoglobin interference detection results of reagents of Example 1, Comparative Examples 1-2 and 5-7

[0209]

[0210] (6) Stability detection

[0211] The reagent kit of the application was subjected to long-term stability test. The reagent kit of the application was calibrated on the test instrument, and was stored in a 2-8℃ environment for 18 months. The serum samples with concentrations of 150 μmol / L and 300 μmol / L were subjected to long-term stability test at 3 months, 6 months, 9 months, 12 months and 18 months, respectively. The deviation value of the test results after opening the bottle for 18 months was calculated, and the results were shown in Table 9.

[0212] Table 9 Stability detection results of Examples 1-5

[0213]

[0214]

[0215] The above merely preferred embodiments of the present application, it should be noted that for those of ordinary skill in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered within the scope of the present application.

Claims

1. A reagent combination for detecting pyruvate, characterized in that, Including reagent 1 and reagent 2; In terms of concentration, reagent 1 comprises: Buffer solution 1 50~100 mM; Stabilizer 20~40 g / L; Surfactant concentration: 20-40 g / L; Preservative 1: 0.1~0.2 g / L; NADH 0.3~0.5 mmol / L; In terms of concentration, reagent 2 comprises: Buffer solution 2 50~100 mM; Preservative 2: 0.5~1.0 g / L; Lactate dehydrogenase 250~400 kU / L; The pH value of reagent 1 is 9.0–10.0; The pH value of reagent 2 is 7.0–8.0; The buffer solution 1 is a CAPSO buffer solution; The buffer solution 2 is a triethanolamine buffer solution; The pH value of the buffer solution 1 is 9.0 to 10.0; The pH value of the buffer solution 2 is 7.0–8.0; The stabilizer is NaCl; The surfactant is GENAPOL® X-080; The preservative 1 or the preservative 2 is independently selected from PC300 and / or NaN3.

2. The reagent combination as described in claim 1, characterized in that, The preservative 1 is PC300; and / or The preservative 2 is NaN3.

3. The reagent combination as described in claim 2, characterized in that, It also includes calibrators; The calibrator was obtained by mixing pure sodium pyruvate with a buffer solution; The concentration of the calibrator includes 200 μmol / L.

4. The use of the reagent combination as described in any one of claims 1 to 3 in the preparation of a pyruvate detection kit.

5. A kit for detecting pyruvate, characterized in that, It includes the reagent combination as described in any one of claims 1 to 3, and acceptable excipients or adjuvants.

6. The reagent kit as described in claim 5, characterized in that, The pH value of the CAPSO is 9.5; and / or The triethanolamine has a pH of 7.

5.

7. The kit according to claim 6, characterized in that, It also includes calibrators; The calibrator was obtained by mixing pure sodium pyruvate with a buffer solution; The concentration of the calibrator includes 200 μmol / L.

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