Cell sialic acid determination method and kit

Abstract

The invention belongs to the technical field of biology, and particularly relates to a cell sialic acid determination method and a kit, and the test kit comprises the following reagents: a cell lysis buffer prepared from NP-40, surfactin and Tris hydrochloric acid; a phosphate matrix buffer solution containing neuraminidase; the enzyme compound prepared from pyruvate oxidase and luciferase contains fluorescein peroxide and a substrate prepared from ATP (adenosine triphosphate). The method is suitable for culturing cell samples, and is simple and convenient to operate, high in sensitivity and good in reagent stability.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method and a kit for determining cell sialic acid (SA). Background Art

[0002] Sialic acid (SA) is a special monosaccharide widely distributed at the end of sugar chains on the cell surface, and its expression level is closely related to a variety of biological and pathological processes. Although sialic acid is widely expressed on the plasma membrane of normal cells, its expression is also upregulated under some pathological conditions, including cancer, infection, neurological diseases, cardiovascular disease (CVD) and diabetic nephropathy. Sialic acid has been identified as a biomarker for cardiovascular disease, diabetes and a range of other inflammatory and degenerative diseases. Therefore, accurate and quantitative detection of cell surface SA is of great significance for clinical diagnosis and treatment.

[0003] The sialic acid detection kits currently sold on the market mostly use colorimetric methods based on chemical reactions or enzymatic methods. The reaction principle of the enzymatic method is: sialic acid α-glycosidic bond is broken by neuraminidase-catalyzed hydrolysis to generate free N-acetylneuraminic acid, N-acetylneuraminic acid is produced into pyruvic acid and mannosamine under the action of neuraminidase aldolase, pyruvic acid and reducing coenzyme (NADH) are produced into lactic acid and NAD+ under the action of lactate dehydrogenase (LDH), and NADH has specific absorption at 340nm. By detecting the change in NADH, the sialic acid content in the blood sample can be detected. This method has the advantages of simple and fast operation and low cost, but low sensitivity. It is suitable for samples with high sialic acid content such as serum and tissue. Customers using existing kits to detect cells are prone to the phenomenon of failure to determine results or low signal values. If the sialic acid content of cells is to be measured, the sialic acid concentration in the cell homogenate supernatant is generally increased by increasing the cell amount or reducing the volume of the homogenate medium. However, there is no guarantee that the sialic acid content of cells can be measured, resulting in an increase in the detection cost.

[0004] The reason is that the sialic acid content of cells cultured in vitro is low, which is lower than the sensitivity of the kit. The sensitivity of the kit needs to be improved to meet the needs of detecting cell samples. Summary of the invention

[0005] In view of this, the present invention improves the existing enzyme method for detecting sialic acid kit to meet the sensitivity requirements of cells, and provides a cell sialic acid luminescence detection kit and method with high sensitivity and simple operation.

[0006] The present invention firstly protects a cell sialic acid luminescence detection kit, comprising a cell lysate, an enzyme complex, and a substrate; wherein:

[0007] The cell lysis solution includes surfactin, tris(hydroxymethyl)aminomethane, NP-40, Triton X-100, and calcium chloride;

[0008] The enzyme complex comprises pyruvate oxidase, thiamine pyrophosphate, flavin adenine dinucleotide, TritonX-405, luciferase, PVP, magnesium chloride, sodium chloride, and di-Gly peptide;

[0009] The substrates include peroxyfluorescein and ATP.

[0010] Specifically, the concentration of tris(hydroxymethyl)aminomethane in the cell lysate is 50 to 200 mmol / L;

[0011] The volume concentration of NP-40 in the cell lysate is 0.5% to 2%;

[0012] The volume concentration of Triton X-100 in the cell lysate is 0.05-0.2%;

[0013] The concentration of calcium chloride in the cell lysate is 5 to 20 mmol / L;

[0014] The volume concentration of the surfactin in the cell lysate is 0.05% to 0.1%;

[0015] The pH of the cell lysis solution is 7.0-8.0.

[0016] Specifically, the concentration of the pyruvate oxidase in the enzyme complex is 500U / L to 1000U / L;

[0017] The concentration of thiamine pyrophosphate in the enzyme complex is 0.5 mmol / L to 2 mmol / L;

[0018] The concentration of the flavin adenine dinucleotide in the enzyme complex is 0.03 to 0.18 mmol / L;

[0019] The volume concentration of Triton X-405 in the enzyme complex is 0.05% to 1.5%;

[0020] The concentration of the luciferase in the enzyme complex is 200U / L to 400U / L;

[0021] The mass concentration of PVP in the enzyme complex is 0.2% to 1%;

[0022] The concentration of magnesium chloride in the enzyme complex is 5 to 15 mmol / L;

[0023] The concentration of sodium chloride in the enzyme complex is 10 to 30 mmol / L,

[0024] The concentration of the di-Gly peptide in the enzyme complex is 50-200 mmol / L.

[0025] Specifically, the structure of the peroxyfluorescein is shown in Formula 3:

[0026]

[0027] The preparation process of the peroxyfluorescein is shown in Formula 1:

[0028]

[0029] The specific reaction process includes:

[0030] Step 1, reacting compound 1 and compound 2 in a 1:1 amount of substance to obtain a solution containing compound 3, the reaction is carried out under the action of a base, the base is selected from 3mmol / L potassium carbonate, the solvent is a mixed solvent of chloroform, water and methanol, the reaction temperature is 25°C to 35°C, the reaction time is 2 to 3h, and a solution containing compound 3 is obtained.

[0031] Step 2, spin-drying to remove chloroform and methanol, adding 1 mol / L hydrochloric acid solution, stirring the mixture for 15 to 20 minutes to form a precipitate, filtering the precipitate, and washing with deionized water until the pH value becomes neutral, which is compound 3;

[0032] Wherein, Formula 1 is peroxy caged fluorescein-2, Formula 2 is N-acetylcysteine, and Formula 3 is peroxyfluorescein.

[0033] Specifically, the concentration of the peroxyfluorescein in the substrate is 0.1-1 g / L; the concentration of the ATP in the substrate is 2-5 g / L.

[0034] Specifically, the cell sialic acid luminescence detection kit further includes a system buffer;

[0035] Specifically, the system buffer comprises phosphate buffer, neuraminidase, N-acetylneuraminic acid aldolase, and enzyme protective agent;

[0036] Specifically, the enzyme protecting agent includes β-cyclodextrin;

[0037] The concentration of the phosphate buffer in the system buffer is 20 mmol / L to 80 mmol / L;

[0038] The concentration of the neuraminidase in the system buffer is 200U / L to 1000U / L;

[0039] The concentration of the N-acetylneuraminic acid aldolase in the system buffer is 1000-5000 U / L;

[0040] The mass concentration of the β-cyclodextrin in the system buffer is 0.5% to 1%;

[0041] The mass concentration of PEG2000 in the system buffer is 0.1% to 0.8%;

[0042] The pH of the system buffer is 6.0-7.0.

[0043] Specifically, the cell sialic acid luminescence detection kit also includes a standard, which is a sialic acid solution, whose solvent is phosphate buffer, the concentration of sialic acid is 1-3 mmol / L, the concentration of phosphate buffer is 20 mmol / L-80 mmol / L and the pH is 6.0-7.0.

[0044] The present invention secondly protects a method for determining the sialic acid content in cells using the above-mentioned cell sialic acid luminescence detection kit, the steps of which are as follows:

[0045] S1. Remove the cell culture medium from the cultured cell sample, add cell lysis solution, let it stand for lysis, and take the cell supernatant after lysis for protein content determination;

[0046] S2, preparing substrate working solution and standard working solutions of different concentrations;

[0047] S3, setting standard wells and assay wells on the ELISA plate, wherein standard working solution is added to the standard wells, and supernatant of lysed cells is added to the assay wells;

[0048] S4, adding system buffer to the standard wells and the assay wells in step S3;

[0049] S5, oscillate to mix, and add enzyme complex after the first incubation;

[0050] S6. Oscillate and mix, add substrate working solution after the second incubation, measure the fluorescence value of each well on a chemiluminescence detector, and determine the content of sialic acid in the cells based on the fluorescence value.

[0051] Calculate the content. The calculation method of cell sialic acid content is as follows:

[0052] Sialic acid content (μmol / gprot) = (△Lb) ÷ a × Cpr × f

[0053] Wherein, △L: the change of the luminescence value of the measured well compared with the luminescence value of the standard well with a concentration of 0; b: the intercept of the standard curve; a: the slope of the standard curve; Cpr: the protein concentration of the sample before adding it to the detection system (gprot / L); f: the dilution factor of the sample before adding it to the detection system.

[0054] Furthermore, the cell sample in step S1 is 0.5 to 1×10 6 The amount of the cell lysis solution is 100-300 μL, and the static lysis time is 5-10 min;

[0055] Step S2: The substrate working solution and the standard working solution are prepared as needed.

[0056] Step S3: The amount of standard working solution and sample used is 10-100 μL;

[0057] The amount of system buffer added in step S4 is 100-150 μL;

[0058] The temperature of the first incubation in step S5 is 35-45° C., the time is 8-16 min, and the amount of the enzyme complex is 20-50 μL;

[0059] The temperature of the second incubation in step S6 is 25-37° C., and the incubation time is 5-12 min;

[0060] Preferably, the reaction temperature in step S5 is 45° C., the incubation is 10 min, and the amount of the enzyme complex is 50 μL.

[0061] Preferably, the reaction temperature in step S6 is 37° C., the incubation time is 15 min, and the amount of substrate used is 50 μL.

[0062] The added volume ratio of the standard, sample, system buffer, enzyme complex and substrate is 2:2:10:5:5.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] (1) The present invention is based on an enzymatic method for detecting sialic acid and adopts a hydrogen peroxide bioluminescence detection system. Compared with the kits on the market that use enzymatic methods to determine absorbance, this kit uses the principle of enzymatic method + chemiluminescence to improve the sensitivity of the reaction, while reducing the amount of cells used and meeting the determination of cell sialic acid content, and is suitable for detecting low-content cell samples. The system also adds an enzyme protective agent to ensure the stability of the kit;

[0065] (2) The present invention provides a lysis solution that can lyse cells without affecting the enzyme activity reaction, without the need for mechanical homogenization, thereby reducing operation requirements, improving sample processing efficiency, simplifying operations, and saving detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0067] Figure 1 This is the reaction principle of the cell sialic acid detection of the present invention;

[0068] Figure 2 is the linearity result of the detection kit in Example 3;

[0069] Figure 3 The figure shows the effects of different β-cyclodextrin and PEG2000 contents on enzyme activity and cell sialic acid (SA) detection results in Example 6. DETAILED DESCRIPTION

[0070] According to a specific embodiment of the present invention, the peroxyfluorescein provided by the present invention is generated by reacting peroxycaged fluorescein-2 (CAS: 1421277-81-7, MV=310) with N-acetylcysteine ​​(CAS: 38520-57-9, MV=162.21).

[0071] The structure of the peroxyfluorescein is shown in Formula 3:

[0072]

[0073] The preparation process of the peroxyfluorescein is shown in Formula 1:

[0074]

[0075] The specific reaction process includes:

[0076] Step 1, compound 1 (Aquila pharmatech, AN15534) and compound 2 (Aladdin, N170064) are reacted in a 1:1 amount of substance to obtain a solution containing compound 3, the reaction is carried out under the action of a base, the base is selected from 3mmol / L potassium carbonate, the solvent is a mixed solvent of chloroform, water and methanol, the reaction temperature is 25°C to 35°C, the reaction time is 2 to 3h, and a solution containing compound 3 is obtained.

[0077] Step 2, spin-drying to remove chloroform and methanol, adding 1 mol / L hydrochloric acid solution, stirring the mixture for 15 to 20 minutes to form a precipitate, filtering the precipitate, and washing with deionized water until the pH value becomes neutral, which is compound 3;

[0078] Wherein, Formula 1 is peroxy caged fluorescein-2, Formula 2 is N-acetylcysteine, and Formula 3 is peroxyfluorescein.

[0079] According to a specific embodiment of the present invention, the reaction principle of the sialic acid detection process is as follows Figure 1 As shown, sialic acid reacts with neuraminidase and N-acetylneuraminic acid acetalase in the system in sequence to generate pyruvic acid, and then pyruvic acid reacts with pyruvate oxidase to generate hydrogen peroxide, which then reacts with peroxyfluorescein to obtain luciferin, which is finally oxidized by luciferase to produce chemiluminescence. The sialic acid content can be measured based on the fluorescence intensity of luciferin.

[0080] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0081] Example 1 Cellular Sialic Acid (SA) Detection Kit

[0082] The following reagents are included:

[0083] Cell lysis solution: weigh 1.211 g tris(hydroxymethyl)aminomethane, 0.11 g calcium chloride, dissolve in 80 mL double distilled water, add 1 ml NP-40 (Aladdin, N105507), 0.1 mL Triton X-100 (Aladdin, T434388), 0.05 mL surfactin (Aladdin, S651680), mix well, adjust the pH to 7.0 with 2 mol / L hydrochloric acid, make up to 100 mL with double distilled water, and store at 4°C.

[0084] System buffer: weigh 2.136g disodium hydrogen phosphate dodecahydrate, 0.5g β-cyclodextrin (Aladdin, C104384), 0.6g PEG2000 (Aladdin, P103723), add 80ml double distilled water to dissolve, adjust the pH to 6.5 with 60mmol / L potassium dihydrogen phosphate solution, and make up to 100ml, that is, 60mmol / L phosphate buffer; take 25mL of 60mmol / L phosphate buffer, add 25U neuraminidase (Aladdin, N755091), 50U N-acetylneuraminic acid aldolase (Sigma, A6680), mix well, and store at 4℃.

[0085] Enzyme complex: Weigh 1.32 g of Gly-2-peptide (Aladdin, G105429), dissolve it in double distilled water, adjust pH to 7.0, make up to 100 mL, take 10 ml of Gly-2-peptide buffer, add 10 U pyruvate oxidase (sigma, P4591), 4.6 mg thiamine pyrophosphate (sigma, T111202), 1 mg flavin adenine dinucleotide (Aladdin, F100614), 0.1 mL of Triton X-405 (Aladdin, T101476), 4 U luciferase (sigma, SRE0045), 0.1 g PVP (Aladdin, P11061), 9.5 mg magnesium chloride, 11 mg sodium chloride, dissolve, and store at 4°C.

[0086] Substrate: Weigh 10 mg of fluorescein peroxygen and 40 mg of ATP (Aladdin, A100880), add 10 mL of double distilled water, dissolve and store at 4°C in the dark.

[0087] Sialic acid standard: Weigh 3.1 mg of sialic acid (Aladdin, A100555), add to pH 6.5, 80 mmol / l phosphate buffer, and store at 4°C.

[0088] Example 2 Cellular Sialic Acid Determination Method

[0089] The method for using the cell sialic acid (SA) detection kit in the above-mentioned Example 1 is as follows:

[0090] (1) Sample processing

[0091] Remove the cell culture medium from the cultured cell samples and press 10 5 Add 100 μL of cell lysis buffer to 10 cells, let stand for 10 minutes, and collect the supernatant. Take part of the supernatant for total protein determination using the BCA method. The cell lysis supernatant needs to be diluted with cell lysis buffer to different concentrations for preliminary experiments.

[0092] (2) Determination

[0093] The detection environment room temperature was 25-30°C, and the final reaction volume was 225 μL.

[0094] The ELISA plate was set up as follows:

[0095] 1 2 3 4 5 6 7 8 9 10 11 12 A A A S1 S9 S17 S25 S33 S41 S49 S57 S65 S73 B B B S2 S10 S18 S26 S34 S42 S50 S58 S66 S74 C C C S3 S11 S19 S27 S35 S43 S51 S59 S67 S75 D D D S4 S12 S20 S28 S36 S44 S52 S60 S68 S76 E E E S5 S13 S21 S29 S37 S45 S53 S61 S69 S77 F F F S6 S14 S22 S30 S38 S46 S54 S62 S70 S78 G G G S7 S15 S23 S31 S39 S47 S55 S63 S71 S79 H H H S8 S16 S24 S32 S40 S48 S56 S64 S72 S80

[0096] Note: AH: standard holes, S1-S80: measurement holes.

[0097] The steps are as follows:

[0098] ① Standard wells: add 20 μL of standard working solution with A and H values ​​of 0 μmol / L, 0.5 μmol / L, 1 μmol / L, 2 μmol / L, 2.5 μmol / L, 3 μmol / L, 4 μmol / L and 5 μmol / L respectively;

[0099] Determination well: add 20 μL of the supernatant of the sample to be tested;

[0100] ③ Add 100 μL of system buffer to each well in step ①;

[0101] ④ Mix by oscillation, incubate at 45°C for 10 min, and then add 50 μL of enzyme complex;

[0102] ⑤ Mix by shaking the plate for 10 seconds, incubate in a 37°C incubator for 15 minutes, and add 50 μL of substrate

[0103] ⑥ Mix by oscillation and detect the luminescence value of each well on a chemiluminescence detector.

[0104] It should be noted that:

[0105] 1. Detection requires the use of a chemiluminescence detector (Luminometer) or a multifunctional microplate reader with chemiluminescence detection function.

[0106] 2. Avoid repeated freezing and thawing of the enzyme complex and store it at -20℃ after aliquoting.

[0107] (3) Calculation of cellular sialic acid (SA) content

[0108] Sialic acid content (μmol / gprot) = (△Lb) ÷ a × Cpr × f

[0109] Wherein, △L: the change of the luminescence value of the measured well compared with the luminescence value of the standard well with a concentration of 0; b: the intercept of the standard curve; a: the slope of the standard curve; Cpr: the protein concentration of the sample before adding it to the detection system (gprot / L); f: the dilution factor of the sample before adding it to the detection system.

[0110] Example 3 Sensitivity Experiment

[0111] According to the kit in Example 1, 1 mmol / L sialic acid standard solution was diluted to 100 μmol / L with cell lysis buffer, and then the luminescence value was determined according to the detection method in Example 2, as follows:

[0112]

[0113] According to the data in the table above, the linear equation is y=19713x+891.34, R 2 =0.9992, draw the standard curve as Figure 2 shown.

[0114] Sensitivity test: With a standard sample volume of 20 μL, the luminescence values ​​of the standard curve and 20 blanks were determined according to the kit operating procedures, as shown in the following table:

[0115] 69 75 78 70 81 68 80 85 65 60 63 81 73 65 67 70 84 72 81 71

[0116] Calculate the standard deviation of the blank value. According to the IUPAC recognized formula, divide 3 times the standard deviation by the slope of the calibration curve to calculate the sensitivity of 0.001μmol / L.

[0117] From the test results, it can be found that the measurement results have a relatively small difference in duplicate wells; and in the reaction system, the linear range of the measured value of the sialic acid standard is good.

[0118] Example 4 Comparison of luciferase detection and peroxidase detection

[0119] Set up 2 experiments, A and B:

[0120] Group A (luciferase assay): assayed according to the cell sialic acid (SA) assay kit of Example 1 and the cell sialic acid assay method of Example 2;

[0121] Group B (peroxidase detection method): The substrate composition is: the hydrogen peroxide substrate is 1.12mmol / L TOOS (a highly sensitive hydrogen peroxide detection substrate in the colorimetric method), 2.5mmol / L 4-AAP, and the luciferase in the enzyme complex is replaced by 20KU / L peroxidase. The other components are the same as the cell sialic acid (SA) detection kit in Example 1. The sample is processed and the reagents are added according to the method of Example 2.

[0122] After the reaction, the cells were detected by an ELISA instrument at a wavelength of 550 nm. Four samples were detected simultaneously: 293T, HepG2, Hela and SHSY5Y cells.

[0123] The test results of Group A are as follows:

[0124] cell Luminous value △L Cpr(g / L) f SA (μmol / gprot) 293T 17215 17003 17037 0.531 1 1.54 HepG2 52215 53003 52537 1.022 2 5.13 Hela 22215 22003 22037 0.724 1 1.48 HS5Y 70382 72494 71365 0.681 1 5.25

[0125] The test results of Group B are as follows:

[0126] cell OD value △OD value f SA (μmol / gprot) 293T 0.043 0.044 0.043 1 0 HepG2 0.045 0.046 0.045 1 0 Hela 0.043 0.045 0.044 1 0 HS5Y 0.044 0.045 0.045 1 0 Blank hole 0.044 0.046 0.045

[0127] Blank wells: Add all reagents but no samples or standards.

[0128] From the above results, we can see that group A can detect the sialic acid content of cells, while all the results of group B are consistent with the results of the blank wells, and it is speculated that its sensitivity is lower. By comparison, it can be seen that under the same conditions, the luciferase system detection method of group A is more sensitive than the peroxidase system detection method of group B.

[0129] Example 5 Effect of surfactin on sialic acid and protein determination

[0130] Set up 3 experiments, A, C and D:

[0131] Group A: cell lysate in the cell sialic acid (SA) detection kit of Example 1;

[0132] Group C: The only difference from Group A is that the cell lysis solution does not contain surfactin, which is a cyclic lipopeptide biosurfactant derived from microorganisms. It can promote calcium ions to pass through the cell membrane and thus promote cell lysis, and will not affect the effects of other components of the reaction system.

[0133] Group D: The only difference from Group A is that SDS was used instead of surfactin.

[0134] Three kits with different cell lysate components were used to simultaneously measure the sialic acid content of four cell samples. The cell dosage was 10 6 The protein content was determined after cell lysis.

[0135] The sialic acid content of the three groups of cells was detected as follows:

[0136]

[0137] The cell protein content of the three groups was detected as follows:

[0138] Sample Type Group A (g / L) Group C (g / L) Group D (g / L) 293T 0.563 0.352 0.571 HepG2 1.233 0.859 1.209 Hela 0.698 0.492 0.702 HS5Y 0.721 0.546 0.719

[0139] From the test results, it can be found that the cell sialic acid value measured in group A is the highest, and the value measured in group C without adding surfactin is lower than that in group A. The total protein content measured in group C is lower, and the degree of cell lysis is lower. The addition of surfactin enhances the lysis effect. The sialic acid content measured in group D is the lowest, but its protein content is the same as that in group A. Because SDS is an anionic surfactant, a common component of cell lysis solution, it has a good lysis effect, but it will inhibit the activity of inhibitory enzymes. The lysis solution with SDS inhibits the enzyme components in the reaction reagent, resulting in low test results. In summary, the addition of surfactin promotes cell lysis and protects the activity of the enzymes in the kit.

[0140] Example 6 Effects of β-cyclodextrin and PEG2000 on enzyme activity and cell sialic acid (SA) detection results

[0141] Set up 4 experiments, A, E, F and G:

[0142] Group A: the system buffer in the cell sialic acid (SA) detection kit of Example 1;

[0143] Group E: The difference from the buffer in group A is that it does not contain β-cyclodextrin and PEG2000;

[0144] Group F: The difference from the buffer in group A is that it contains β-cyclodextrin but no PEG2000;

[0145] Group G: The difference from the buffer solution in Group A is that it does not contain β-cyclodextrin but contains PEG2000.

[0146] The above 4 groups of system buffers were placed in a 4°C refrigerator, and quality control products with a target value of 4.5±0.2 μmol / L were detected weekly using a cell sialic acid (SA) detection kit containing the above 4 system buffers and other components being the same as in Example 1, and the same determination method as in Example 2, and changes in the determination values ​​of the quality control products were monitored.

[0147] The system buffer provides the enzyme and buffer environment for hydrolyzing sialic acid to produce pyruvic acid. The content of pyruvic acid produced determines the sensitivity of the kit. The activity of the enzyme is the key factor. Figure 3 It can be seen that the addition of β-cyclodextrin and PEG2000 protects the enzyme activity and increases the stability of the reagent. From the above results, it is found that only when β-cyclodextrin and PEG2000 are added at the same time can the corresponding effect be achieved.

[0148] Example 7 Effect of Triton X-405 on enzyme stability and cell sialic acid (SA) detection results

[0149] Set up 4 experiments, A, H, I and J:

[0150] Group A: the enzyme complex in the cell sialic acid (SA) detection kit of Example 1 (wherein the concentration of Trition X-405 is 0.1 mL / 100 mL, and the concentration of PVP is 0.1 g / 100 mL);

[0151] Group H: The enzyme complex differs from Group A only in that it does not contain Triton X-405 and PVP;

[0152] Group I: The enzyme complex differed from Group A only in that the concentration of Trition X-405 was 0.05% and the concentration of PVP was 0.1%;

[0153] Group J: The enzyme complex differed from that in Group A only in that the concentration of Trition X-405 was 2% and the concentration of PVP was 0.5%;

[0154] The above four groups of enzyme complexes were placed in a 4°C refrigerator, and the quality control products with a target value of 3.5±0.2 μmol / L were detected weekly using a cellular sialic acid (SA) detection kit containing the above four enzyme complexes and other components being the same as those in Example 1, and the same determination method as in Example 2, and the changes in the determination values ​​of the quality control products were monitored.

[0155]

[0156]

[0157] From the above results, it can be seen that the addition of Triton X-405 and PVP is beneficial to the stability of the kit, but their concentrations need to be within a certain range. Too much Triton X-405 or too little PVP cannot achieve a stabilizing effect, and they need to be in a suitable ratio.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A cell sialic acid luminescence detection kit, characterized in that: Contains cell lysate, enzyme complex, substrate; in, The cell lysis solution includes surfactin, tris(hydroxymethyl)aminomethane, NP-40, Triton X-100, and calcium chloride; The enzyme complex comprises pyruvate oxidase, thiamine pyrophosphate, flavin adenine dinucleotide, Triton X-405, luciferase, PVP, magnesium chloride, sodium chloride, and di-Gly peptide; The substrates include peroxyfluorescein and ATP.

2. The cell sialic acid luminescence detection kit according to claim 1, characterized in that: The concentration of tris(hydroxymethyl)aminomethane in the cell lysate is 50-200 mmol / L; The volume concentration of NP-40 in the cell lysate is 0.5% to 2%; The volume concentration of Triton X-100 in the cell lysate is 0.05-0.2%; The concentration of calcium chloride in the cell lysate is 5 to 20 mmol / L; The volume concentration of the surfactin in the cell lysate is 0.05% to 0.1%; The pH of the cell lysis solution is 7.0-8.

0.

3. The cell sialic acid luminescence detection kit according to claim 1, characterized in that: The concentration of the pyruvate oxidase in the enzyme complex is 500U / L to 1000U / L; The concentration of thiamine pyrophosphate in the enzyme complex is 0.5 mmol / L to 2 mmol / L; The concentration of the flavin adenine dinucleotide in the enzyme complex is 0.03 to 0.18 mmol / L; The volume concentration of Triton X-405 in the enzyme complex is 0.05% to 1.5%; The concentration of the luciferase in the enzyme complex is 200U / L to 400U / L; The mass concentration of PVP in the enzyme complex is 0.2% to 1%; The concentration of magnesium chloride in the enzyme complex is 5 to 15 mmol / L; The concentration of sodium chloride in the enzyme complex is 10 to 30 mmol / L, The concentration of the di-Gly peptide in the enzyme complex is 50-200 mmol / L.

4. The cell sialic acid luminescence detection kit according to claim 1, characterized in that: The structure of the peroxyfluorescein is shown in Formula 3:

5. The cell sialic acid luminescence detection kit according to claim 1, characterized in that: The concentration of the peroxyfluorescein in the substrate is 0.1 to 1 g / L; The concentration of the ATP in the substrate is 2-5 g / L.

6. The cell sialic acid luminescence detection kit according to claim 1, characterized in that: The cell sialic acid luminescence detection kit also includes a system buffer.

7. The cell sialic acid luminescence detection kit according to claim 6, characterized in that: The system buffer comprises phosphate buffer, neuraminidase, N-acetylneuraminic acid aldolase and enzyme protective agent.

8. The cell sialic acid luminescence detection kit according to claim 7, characterized in that: The enzyme protecting agent includes β-cyclodextrin; The concentration of the phosphate buffer in the system buffer is 20 mmol / L to 80 mmol / L; The concentration of the neuraminidase in the system buffer is 200U / L to 1000U / L; The concentration of the N-acetylneuraminic acid aldolase in the system buffer is 1000-5000 U / L; The mass concentration of the β-cyclodextrin in the system buffer is 0.5% to 1%; The mass concentration of PEG2000 in the system buffer is 0.1% to 0.8%; The pH of the system buffer is 6.0-7.

0.

9. A method for measuring the sialic acid content in cells using the cell sialic acid luminescence detection kit according to any one of claims 1 to 8, characterized in that: Here are the steps: S1. Remove the cell culture medium from the cultured cell sample, add cell lysis solution, let it stand for lysis, and take the cell supernatant after lysis for protein content determination; S2, preparing substrate working solution and standard working solutions of different concentrations; S3, setting standard wells and assay wells on the ELISA plate, wherein standard working solution is added to the standard wells, and supernatant of lysed cells is added to the assay wells; S4, adding system buffer to the standard wells and the assay wells in step S3; S5, oscillate to mix, and add enzyme complex after the first incubation; S6. Oscillate and mix, add substrate working solution after the second incubation, measure the fluorescence value of each well on a chemiluminescence detector, and determine the content of sialic acid in the cells based on the fluorescence value.

10. The method according to claim 9, characterized in that The cell sample in step S1 is 0.5 to 1×10 6 The amount of the cell lysis solution is 100-300 μL, and the static lysis time is 5-10 min; Step S3: The amount of standard working solution and sample used is 10-100 μL; The amount of system buffer added in step S4 is 100-150 μL; The temperature of the first incubation in step S5 is 35-45° C., the time is 8-16 min, and the amount of the enzyme complex is 20-50 μL; The temperature of the second incubation in step S6 is 25-37° C., and the incubation time is 5-12 min; The added volume ratio of the standard, sample, system buffer, enzyme complex and substrate is 2:2:10:5:5.

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