A cell lysate, an enzyme activity detection kit for cells and applications thereof

Abstract

The present invention discloses a cell lysate, an enzyme activity detection kit for cells and applications thereof. The components of the cell lysate include: a first buffer, an isotonic component, Triton X-100, sodium deoxycholate, EDTA, BSA, sucrose, glycerol, β-cyclodextrin, mannitol, hexose, sodium carboxymethyl cellulose, sodium sulfate, dextran, PVP, glycine, PEG6000. The cell lysate of the present invention removes the ionic detergent component SDS, avoiding the denaturation and inactivation of the enzymes released by cell lysis due to the strong denaturing effect of SDS. At the same time, the combination of Triton X-100 and sodium deoxycholate can meet the requirement of destroying cell membranes and organelle membranes. On the other hand, β-cyclodextrin, sodium carboxymethyl cellulose, dextran, etc. that protect enzyme activity are added to protect the stability of the enzymes released by cells after cell lysis and the enzyme structure in the kit. The cell lysate of the present invention is suitable for the detection of enzyme activity in cells, reducing the difficulty of sample processing.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme activity detection, and particularly relates to a cell lysate, a kit for detecting enzyme activity in cells and an application thereof. Background Art

[0002] Enzyme activity detection plays an irreplaceable role in disease research and drug development. Clinically, it is generally to detect enzyme activity in human serum, saliva, milk, urine, feces, etc., or tissues to determine whether the body is normal; when developing drugs, mice and rats are used to repeat the enzyme activity detection in the above samples. These detection studies are all evaluated at the macroscopic level of the organism. With the development of science and technology and people's attention to health, the research at the cellular level of the organism has been continuously deepened, and cell metabolism research has become one of the hottest research topics. Enzyme activity detection is part of metabolic detection, and cell enzyme activity detection is part of cell metabolism detection. The research on cell enzyme activity is inseparable from the related technologies and supporting reagents for cell metabolism detection. With the in-depth study of cells, the demand is getting larger and higher. They belong to an important branch of cell detection. In order to more intuitively show cell functions and states, it is necessary to innovate traditional cell metabolism detection technologies and reagents.

[0003] For the existing cell enzyme activity detection, generally, cells are homogenized at low temperature or sonicated with an isotonic solution, and then after collecting the homogenate, it is centrifuged at high speed, and the supernatant is taken as the detection object. The operation is complex, time-consuming and laborious, which is particularly obvious when there are many samples. On the other hand, in order to ensure detection, a relatively large amount of cells is used, increasing the usage cost. When detecting cell enzyme activity, using mechanical homogenization or sonication cannot completely extract the enzyme. Although the traditional lysis buffer RIPA has a high extraction efficiency, its lysis characteristics will have an obvious impact on enzyme activity and the detection system, so it is not applicable.

[0004] Therefore, it is particularly important to provide a cell lysate that can efficiently process cells, increase the cell breakage rate, and reduce the cell usage amount to improve the overall experimental efficiency. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above technical deficiencies, and propose a cell lysate, a kit for detecting enzyme activity in cells and an application thereof, to solve the technical problems in the prior art that during the detection of various enzyme activities in cells, the enzyme activity extracted with the traditional lysis buffer is reduced, the sample processing with mechanical homogenization is complex, and the number of samples that can be processed is limited.

[0006] In a first aspect, the present invention provides a cell lysate, and the components of the cell lysate include: 25 - 150 mM of a first buffer, 75 - 125 mM of an isotonic component, 1% Triton X-100, 0.25% sodium deoxycholate, 1 - 2 mmol / L of EDTA, 0.1 - 0.5% BSA, 20 - 80 g / L of sucrose, 1 - 3% glycerol, 0.1 - 0.5% β-cyclodextrin, 10 - 20 g / L of mannitol, 10 - 20 g / L of hexose, 0.1 - 0.5% sodium carboxymethyl cellulose, 10 - 25 mM of sodium sulfate, 0.1 - 0.5% dextran, 0.5 - 1.5% PVP, 5 - 10 mM of glycine, 1 - 5% PEG6000.

[0007] In a second aspect, the present invention provides a kit for detecting enzyme activity in cells, and the kit for detecting enzyme activity in cells includes the cell lysate provided in the first aspect of the present invention.

[0008] In a third aspect, the present invention provides the application of the above kit for detecting enzyme activity in cells in detecting enzyme activity in cells.

[0009] Compared with the prior art, the beneficial effects of the present invention include:

[0010] Compared with a common RIPA lysate, the cell lysate of the present invention removes the ionic detergent component SDS, avoiding the denaturation and inactivation of the enzymes released by cell lysis due to the strong denaturing effect of SDS. At the same time, the combination of Triton X-100 and sodium deoxycholate can already meet the requirement of destroying cell membranes and organelle membranes. On the other hand, some components such as β-cyclodextrin, sodium carboxymethyl cellulose, and dextran that protect enzyme activity are added to protect the stability of the enzymes released from cells and the enzyme structure in the kit after cell lysis. The cell lysate of the present invention is suitable for detecting the activity of various enzymes in cells, reducing the difficulty of sample processing, and more samples can be processed within the same time using the lysate of the present invention. Using the above cell lysate to prepare a kit for detecting enzyme activity in cells based on immobilization technology can protect the enzyme activity in the sample, and the components in the lysate can also protect the enzymes in the kit. On the other hand, it reduces the difficulty of detecting cell samples and improves the detection efficiency of cell samples. Description of the Drawings

[0011] Figure 1 They are micrographs of cells treated in different ways; A is the cells treated with the ALTD cell lysate, B is the cells treated by mechanical homogenization, and C is the cells treated with the RIPA lysate;

[0012] Figure 2 It is a comparison chart of the glucose oxidase enzyme activity in HL-60 cells treated with the ALTD cell lysate of the present invention after serum starvation for 12 h and treated with mannitol-containing methanol serum for 12 h;

[0013] Figure 3 It is a comparison chart of the enzyme activity of lactate dehydrogenase in HL-60 cells treated with the ALTD cell lysate of the present invention after being normally placed for 1 h and treated with carbon tetrachloride for 1 h;

[0014] Figure 4 It is a comparison chart of the enzyme activity of glucose oxidase in HL-60 cells treated with the ALTD cell lysate of the present invention and the traditional RIPA lysate;

[0015] Figure 5 After the ALTD cell lysate of the present invention and the traditional RIPA lysate are processed and then left for 10 min, 20 min, and 30 min, the enzyme activities of three enzymes, namely glucose oxidase, lactate dehydrogenase, and alkaline phosphatase, extracted from HL-60 cells are detected;

[0016] Figure 6 It is a standard curve for detecting the enzyme activity of glucose oxidase in cells with different lysates; among them, the abscissa is the reciprocal of the sample dilution factor, and the ordinate is the detected OD value; y = 2.7711x + 0.0138 is the standard curve for the combination of the cell lysate of the present invention and the kit, and y = 0.39x + 0.0335 is the standard curve for the combination of RIPA lysate and the commercially available kit. Detailed implementation manners

[0017] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0018] In a first aspect, the present invention provides a cell lysate (named ALTD cell lysate), and the components of the cell lysate include: 25 - 150 mM of a first buffer, 75 - 125 mM of an isotonic component, 1% Triton X-100, 0.25% sodium deoxycholate, 1 - 2 mM of EDTA (ethylenediaminetetraacetic acid), 0.1 - 0.5% of BSA (bovine serum albumin), 20 - 80 g / L of sucrose, 1 - 3% of glycerol, 0.1 - 0.5% of β-cyclodextrin, 10 - 20 g / L of mannitol, 10 - 20 g / L of hexose, 0.1 - 0.5% of sodium carboxymethyl cellulose, 10 - 25 mM of sodium sulfate, 0.1 - 0.5% of dextran, 0.5 - 1.5% of PVP (polyvinylpyrrolidone), 5 - 10 mM of glycine, and 1 - 5% of PEG6000. In the formula of the present invention, "%" refers to the mass fraction.

[0019] In the cell lysate formula of the present invention, 1% Triton X-100 and 0.25% sodium deoxycholate are used to lyse cell membranes and organelle membranes; 1-2 mM EDTA, 0.1-0.5% BSA, 20-80 g / L sucrose, 1-3% glycerol, 10-20 g / L hexose, 10-25 mM sodium sulfate, and 5-10 mM glycine are enzyme activity protectants; 0.1-0.5% β-cyclodextrin, 10-20 g / L mannitol, 0.1-0.5% sodium carboxymethyl cellulose, 0.1-0.5% dextran, 0.5-1.5% PVP, and 1-5% PEG6000 are enzyme structure stabilizers.

[0020] In this embodiment, the first buffer is Tris-HCl with a pH of 7-8, further 7.4.

[0021] In this embodiment, the isotonic component is NaCl.

[0022] In a second aspect, the present invention provides a kit for detecting enzyme activity in cells, which includes the cell lysate provided in the first aspect of the present invention.

[0023] In this embodiment, the above-mentioned kit for detecting enzyme activity in cells is a kit for detecting enzyme activity in cells based on a fixation technique.

[0024] The present invention uses a kit for detecting enzyme activity in cells based on a fixation technique to immobilize the substrate, or the substrate and the enzyme, in an enzyme-linked immunosorbent assay (ELISA) plate, reducing the reagent composition, extending the reagent storage time, improving the detection efficiency, and reducing the use cost.

[0025] Further, in the kit for detecting enzyme activity in cells based on this fixation technique, when detecting, for every 10 5 -10 6 cells, 100-200 μL of the cell lysate of the present invention is added.

[0026] Further, the kit for detecting enzyme activity in cells based on this fixation technique includes:

[0027] An ELISA plate coated with a substrate for detecting enzyme activity in cells, or a substrate plus an enzyme; the present invention places no restrictions on the substrate and enzyme in the ELISA plate coated, and those skilled in the art can make selections according to actual situations. For example, when detecting glucose oxidase, glucose and horseradish peroxidase are coated on the ELISA plate; when detecting lactate oxidase, lactate and horseradish peroxidase are coated on the ELISA plate; when detecting lactate dehydrogenase, lactate and coenzyme I are coated on the ELISA plate; when detecting alkaline phosphatase, disodium p-nitrophenyl phosphate and magnesium chloride are coated on the ELISA plate, etc.

[0028] Enzyme activity detection chromogenic reagent, which is used to detect the enzyme in cells and the substrate in the coated enzyme-linked immunosorbent assay (ELISA) plate, or the product generated by the enzymatic reaction of the substrate plus enzyme, or the consumed substrate; the present invention does not limit the enzyme activity detection chromogenic reagent, and those skilled in the art can select according to the actual situation. For example, when detecting glucose oxidase or lactate oxidase, the enzyme activity detection chromogenic reagent contains chromogenic substances such as 4-aminoantipyrine and phenol, chlorophenol or TOOS; when detecting lactate dehydrogenase, the enzyme activity detection chromogenic reagent contains 2,4-dinitrophenylhydrazine and sodium hydroxide; when detecting alkaline phosphatase, the enzyme activity detection chromogenic reagent contains 2-amino-2-methyl-1-propanol or diethanolamine.

[0029] Standard, a reference for measuring enzyme activity, which may not be used in some enzyme activity detection processes; the present invention does not limit the standard, and those skilled in the art can select according to the actual situation. For example, when detecting glucose oxidase or lactate oxidase, the standard is hydrogen peroxide, and when detecting other enzymes, the standard can be an enzyme, a product or a substrate; when detecting lactate dehydrogenase, the standard is sodium pyruvate; when detecting alkaline phosphatase, the standard is p-nitrophenol.

[0030] Cell lysate, which is used to lyse cells to obtain enzymes, and the cell lysate is the cell lysate provided in the first aspect of the present invention.

[0031] The present invention also does not limit the quantity and type of the coated ELISA plate, enzyme activity detection chromogenic reagent, and standard in the cell enzyme activity detection kit. Those skilled in the art can select according to the actual situation. In some specific embodiments of the present invention, the above-mentioned coated ELISA plate includes the same or different coated ELISA plate 1, coated ELISA plate 2, coated ELISA plate 3, coated ELISA plate 4,..., coated ELISA plate N, which are used to coat the same or different substrates, or the same or different substrates and enzymes; the above-mentioned enzyme activity detection chromogenic reagent includes the same or different enzyme activity detection chromogenic reagent 1, enzyme activity detection chromogenic reagent 2, enzyme activity detection chromogenic reagent 3, enzyme activity detection chromogenic reagent 4,..., enzyme activity detection chromogenic reagent N, which are used to detect the same or different enzymes in cells; the above-mentioned calibrator includes the same or different standards 1, calibrators 2, calibrators 3, calibrators 4,..., calibrators N, which are used to measure the same or different enzymes in cells.

[0032] In the third aspect, the present invention provides the application of the above-mentioned cell enzyme activity detection kit in detecting the enzyme activity in cells.

[0033] In this embodiment, the above-mentioned cell enzyme activity detection kit is a cell enzyme activity detection kit based on immobilization technology, and the above application includes the following steps:

[0034] S1. Sample preparation: Collect 10 5 -10 6A cell sample to be tested is washed thoroughly with pre-cooled second buffer to wash away components such as culture medium;

[0035] S2. Sample treatment: After adding 100 - 200 μL of the cell lysate provided in the first aspect of the present invention to the collected cells, mix well and place on ice for lysis, centrifuge to obtain the supernatant, and obtain the sample to be tested;

[0036] S3. Standard preparation: Dilute with cell lysate to obtain a series of standard solutions with different concentrations;

[0037] S4. Measurement: Set standard wells and measurement wells on the coated enzyme-linked immunosorbent assay (ELISA) plate, add V 1 volumes of a series of standard solutions with different concentrations to the standard wells, add V 1 volumes of the sample to be tested to the measurement wells, then add V 2 volumes of the enzyme activity detection chromogenic reagent to the standard wells and measurement wells, mix well and incubate at temperature T, and detect the OD values A 1 and A 2 at two fixed time points at the wavelength of λ nm of the ELISA reader, and calculate the change value ΔA;

[0038] S5. Draw a standard curve and calculate the enzyme activity through the general cell enzyme activity formula shown in formula (1); where formula (1) is as follows:

[0039] X enzyme activity (U / gprot) = (ΔA - b) ÷ a ÷ C pr ÷ t × f (1);

[0040] In formula (1), a is the slope of the standard curve, b is the intercept of the standard curve, t is the interval time between two fixed time points in minutes; C pr is the protein content in the sample to be tested after centrifugation in step S2; f is the dilution factor of the sample to be tested after centrifugation before adding to the ELISA plate wells in the sample treatment step; the definition of enzyme activity in formula (1) is: at temperature T, the amount of enzyme that catalyzes the production of 1 μmol of product per gram of protein per minute is 1 activity unit; or the definition of enzyme activity in formula (1) is: at temperature T, the amount of enzyme that catalyzes the consumption of 1 μmol of substrate per gram of protein per minute is 1 activity unit.

[0041] In this embodiment, in step S1, the second buffer is phosphate buffer solution (PBS).

[0042] Further, the concentration of the phosphate buffer solution is 0.005 - 0.02 M, more preferably 0.01 M, pH = 7 - 8, more preferably 7.4.

[0043] In this embodiment, in step S1, the number of washing times is 2 - 5 times, further 3 times.

[0044] In this embodiment, in step S2, the lysis temperature is 0 - 8 °C, the lysis time is 5 - 40 min, preferably 8 - 12 min.

[0045] In this embodiment, step S2 specifically includes: adding the cell lysate provided in the first aspect of the present invention to the collected cells, mixing on a vortex mixer for 3 - 5 seconds, placing on ice, mixing once every 3 - 5 min, then lysing on ice, and subsequently centrifuging at 9000 - 10000×g at 4 °C for 8 - 12 min, taking the supernatant to obtain the sample to be tested.

[0046] In this embodiment, in step S4, V 1 is 1 - 250 μL, V 2 is 50 - 299 μL, the temperature T is 32 - 40 °C, the detection wavelength λ is 260 - 800 nm, and the change value ΔA is |A 1 -A 2 |.

[0047] In this embodiment, in step S5, t in formula (1) is 0.5 - 120.

[0048] Example 1

[0049] In this example, the specific components of the ALTD cell lysate are: 25 mM Tris-HCl (pH = 7.4), 75 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EDTA, 0.1% BSA, 20 g / L sucrose, 1% glycerol, 0.1% β-cyclodextrin, 10 g / L mannitol, 10 g / L hexose, 0.1% sodium carboxymethyl cellulose, 10 mM sodium sulfate, 0.1% dextran, 0.5% PVP, 5 mM glycine, 1% PEG6000.

[0050] Example 2

[0051] In this example, the specific components of the ALTD cell lysate are: 75 mM Tris-HCl (pH = 7.4), 100 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 1.5 mM EDTA, 0.25% BSA, 50 g / L sucrose, 1.5% glycerol, 0.25% β-cyclodextrin, 15 g / L mannitol, 15 g / L hexose, 0.25% sodium carboxymethyl cellulose, 15 mM sodium sulfate, 0.25% dextran, 1% PVP, 7.5 mM glycine, 2.5% PEG6000.

[0052] Example 3

[0053] In this example, the specific components of the ALTD cell lysate are: 150 mM Tris-HCl (pH = 7.4), 125 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 2 mM EDTA, 0.5% BSA, 80 g / L sucrose, 3% glycerol, 0.5% β-cyclodextrin, 20 g / L mannitol, 20 g / L hexose, 0.5% sodium carboxymethyl cellulose, 25 mM sodium sulfate, 0.5% dextran, 1.5% PVP, 10 mM glycine, 5% PEG6000.

[0054] Example 4

[0055] This example provides a kit for detecting enzyme activity in fixed cells, which includes:

[0056] A coated enzyme-linked immunosorbent assay (ELISA) plate, which is used to provide the substrate for detecting enzyme activity in cells, or the substrate plus enzyme. For detecting glucose oxidase, 0.5 mmol of glucose and 5 U of horseradish peroxidase are coated in the coated ELISA plate; for detecting lactate oxidase, 0.1 mmol of lactate and 5 U of horseradish peroxidase are coated in the wells of the coated ELISA plate; for detecting lactate dehydrogenase, 0.2 mmol of lactate and 0.1 mmol of coenzyme I are coated in the wells of the coated ELISA plate; for detecting alkaline phosphatase, 0.5 mmol of disodium 4-nitrophenyl phosphate and 0.02 mmol of magnesium chloride are coated in the wells of the coated ELISA plate.

[0057] An enzyme activity detection color reagent, which is used to detect the product generated or the substrate consumed by the enzymatic reaction of the enzyme in cells with the substrate, or the substrate plus enzyme, in the coated ELISA plate. For detecting glucose oxidase or lactate oxidase, the enzyme activity detection color reagent contains 2 mmol / L 4-aminoantipyrine and 5 mmol / L phenol; for detecting lactate dehydrogenase, the enzyme activity detection color reagent contains 4 mmol / L 2,4-dinitrophenylhydrazine and 1 mol / L sodium hydroxide; for detecting alkaline phosphatase, the enzyme activity detection color reagent contains 1 mol / L 2-amino-2-methyl-1-propanol.

[0058] A standard product, which is a reference for measuring enzyme activity. For detecting glucose oxidase or lactate oxidase, the standard product is hydrogen peroxide solutions at 8 concentrations of 0, 0.2, 0.3, 0.4, 0.6, 0.8, 0.9, 1 mmol / L; for detecting lactate dehydrogenase, the standard product is sodium pyruvate solutions at 8 concentrations of 0, 0.2, 0.4, 0.8, 1.2, 1.6, 1.8, 2 mmol / L; for detecting alkaline phosphatase, the standard product is p-nitrophenol solutions at 8 concentrations of 0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5 g / L.

[0059] A lysate, which is used to lyse cells to obtain enzymes, and the lysate is any of the lysates for detecting enzyme activity in cells in Examples 1-3.

[0060] Example 5

[0061] This embodiment provides the application of the cell enzyme activity detection kit based on the immobilization technology in Example 4 in detecting the enzyme activity in cells. Specifically, it includes the following steps:

[0062] (1) Sample preparation: Collect 10 6 Centrifuge the cells at 4°C, 500×g in a refrigerated centrifuge, remove the supernatant, add 1 mL of pre-cooled PBS (0.01 M, pH=7.4) to wash thoroughly, and centrifuge at 4°C, 500×g in a refrigerated centrifuge to remove the supernatant (it is generally recommended to wash 3 times with 1 mL of pre-cooled PBS).

[0063] (2) Sample processing: Add 200 μL of cell lysis buffer to the collected cells and mix on a vortex mixer for 5 seconds. Place on ice, mix once every 5 minutes, lyse on ice for 10 minutes, centrifuge at 10,000 × g for 10 minutes at 4°C, and collect the supernatant to obtain the sample to be tested.

[0064] It should be noted that for the samples to be tested after cell sample processing, the total protein concentration (C pr ), specifically, the BCA method can be used to measure the protein concentration.

[0065] (3) Preparation of standard: Dilute with cell lysis buffer to obtain a series of standard solutions with different concentrations.

[0066] (4) Determination:

[0067] Table 1 ELISA plate settings

[0068] 1 2 3 4 5 6 7 8 9 10 11 12 A A1 A1 S1 S9 S17 S25 S33 S41 S49 S57 S65 S73 B B2 B2 S2 S10 S18 S26 S34 S42 S50 S58 S66 S74 C C3 C3 S3 S11 S19 S27 S35 S43 S51 S59 S67 S75 D D4 D4 S4 S12 S20 S28 S36 S44 S52 S60 S68 S76 E E5 E5 S5 S13 S21 S29 S37 S45 S53 S61 S69 S77 F F6 F6 S6 S14 S22 S30 S38 S46 S54 S62 S70 S78 G G7 G7 S7 S15 S23 S31 S39 S47 S55 S63 S71 S79 H H8 H8 S8 S16 S24 S32 S40 S48 S56 S64 S72 S80

[0069] The specific operations are as follows:

[0070] ① According to the setting method in Table 1, add 100 μL of a series of standard solutions of different concentrations to the corresponding standard wells in the ELISA plate;

[0071] ② Add 100 μL of the sample to be tested to the corresponding measurement wells in the ELISA plate;

[0072] ③ Add 200 μL of enzyme activity detection colorimetric reagent to the standard wells and assay wells in steps ① and ②, and shake the plate on the microplate reader to mix well;

[0073] ④ Incubate at 37°С, and record the OD value at 0min and 10min at λnm wavelength using a microplate reader as A. 1 , A 2 , and calculate the change value △A=|A 1-A 2 |;

[0074] It should be noted that when using a pipette to transfer liquid, be careful to avoid bubbles. During the test, the ambient room temperature is 25°C;

[0075] (5) Draw a standard curve and calculate the enzyme activity by the enzyme activity in the universal cell shown in formula (1); formula (1) is as follows:

[0076] X enzyme activity (U / gprot) = (△Ab) ÷ a ÷ C pr ÷t×f(1)

[0077] In formula (1), a is the slope of the standard curve, b is the intercept of the standard curve, and t is the interval between two fixed time points, 10 minutes; C pr is the protein concentration of the supernatant after centrifugation in the sample processing step; f is the dilution multiple of the supernatant after centrifugation in the sample processing step before adding it to the well of the ELISA plate; the enzyme activity in formula (1) is defined as: at a temperature of 37°C, the amount of enzyme that catalyzes the production of 1 μmol of product per gram of protein per minute is 1 activity unit; or the enzyme activity in formula (1) is defined as: at a temperature of 37°C, the amount of enzyme that catalyzes the consumption of 1 μmol of substrate per gram of protein per minute is 1 activity unit;

[0078] Wherein, in step (4), the detection wavelength λ is 260-800nm.

[0079] Application Example 1

[0080] The cells were lysed using the ALTD cell lysate in Example 1 combined with the kit in Example 4 and the method in Example 5; the cells were mechanically homogenized with PBS (10 6 The cell samples to be tested were centrifuged at 4°C, 500×g centrifugal force, after removing the cell supernatant, 1 mL of pre-cooled PBS (0.01M, pH=7.4) was added for thorough washing, and the supernatant was removed by centrifuging at 4°C, 500×g centrifugal force, repeated 3 times, and then 0.2 mL of pre-cooled PBS and a steel ball were added, and the samples were placed in a mechanical homogenizer, and the parameters of 45 Hz, 60 s, and 4°C were set for homogenization); the cells were treated with RIPA lysis buffer (50 mM Tris-HCl (pH7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS) (10 cells were collected) 6For a cell sample to be tested, after removing the cell supernatant using a refrigerated centrifuge at 4°C and a centrifugal force of 500×g, add 1 mL of pre-cooled PBS (0.01 M, pH = 7.4) and wash thoroughly. Then, remove the supernatant using a refrigerated centrifuge at 4°C and a centrifugal force of 500×g, and repeat this process 3 times. Next, add 0.2 mL of RIPA lysis buffer, mix well on a vortex mixer for 5 seconds, place on ice, mix once every 5 minutes, and lyse on ice for 10 minutes. Observe the cells treated in these three ways under a microscope, and the results are shown in Figure 1 , where A is the cells treated with ALTD cell lysate; B is the cells treated by mechanical homogenization; C is the cells treated with RIPA lysis buffer.

[0081] From Figure 1 The results show that when using the ALTD cell lysate prepared by the present invention to treat cells, the cells are completely broken, and the macromolecules are intact; when treating cells by mechanical homogenization, there is less cell breakage; when treating cells with RIPA lysis buffer, the cells are completely broken, but over-lysed, resulting in the destruction of enzyme macromolecules. It can be seen that the cell lysate of the present invention can completely break cells to release enzyme molecules, and at the same time can protect the enzyme structure and avoid inactivation.

[0082] Application Example 2

[0083] Use the ALTD cell lysate of Example 2 in combination with the kit in Example 4 and the detection method in Example 5 to detect the glucose oxidase activity in HL-60 cells. Among them:

[0084] During detection, the detection wavelength λ is 550 nm.

[0085] Application Example 3

[0086] Carry out in the same way as Application Example 1, except that HL-60 is replaced with THP-1 suspension cells.

[0087] Application Example 4

[0088] Carry out in the same way as Application Example 1, except that HL-60 is replaced with HepG2 adherent cells.

[0089] Application Example 5

[0090] Use the ALTD cell lysate in Example 3 to treat HL-60 cells that have been serum-starved for 12 h and treated with mannitol and methanol-containing serum for 12 h respectively, and then carry out according to the kit in Example 4 and the detection method in Example 5 to detect the glucose oxidase activity. During detection, the detection wavelength λ is 550 nm, and the results are shown in Figure 2 .

[0091] From Figure 2The results showed that the ALTD cell lysate prepared by the present invention can well extract glucose oxidase from HL-60 cells that have been serum starved for 12 hours and treated with mannitol-methanol serum for 12 hours; the detection results showed obvious differences, indicating that the ALTD cell lysate of the present invention exhibits excellent lysis ability for cell treatment under different conditions.

[0092] Application Example 5

[0093] The HL-60 cells were placed normally for 1 hour and treated with carbon tetrachloride for 1 hour using the ALTD cell lysate in Example 3, and then the lactate dehydrogenase activity was detected according to the kit in Example 4 and the detection method in Example 5. During the detection, the detection wavelength λ was 450nm. The results are shown in FIG. Figure 3 .

[0094] Depend on Figure 3 The results showed that the ALTD cell lysate prepared in Example 3 of the present invention can well extract lactate dehydrogenase from HL-60 cells that have been left normally for 1 hour and treated with carbon tetrachloride for 1 hour, and the detection results showed obvious differences, indicating that the lysate of the present invention can have a good effect on cell treatment under different conditions, and can also detect the activity of different types of enzymes, showing good practicality and simplicity.

[0095] Comparative Example 1

[0096] Glucose oxidase in HL-60 cells was extracted using the ALTD cell lysate in Example 1 and a conventional RIPA lysate (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS), respectively. Then, the activity of glucose oxidase was detected according to the kit in Example 4 and the detection method in Example 5. During the detection, the detection wavelength λ was 550 nm. The results are shown in FIG. Figure 4 .

[0097] Depend on Figure 4 It can be seen that the glucose oxidase activity detected after extraction with the ALTD lysis solution in Example 1 is high, while the glucose oxidase activity detected after extraction with the traditional RIPA lysis solution is low, indicating that the ALTD cell lysis solution of the present invention has a better effect on the lysis and extraction of glucose oxidase in HL-60 cells than the traditional RIPA lysis solution.

[0098] Comparative Example 2

[0099] According to the kit in Example 4 and the detection method in Example 5, 3 portions of 10 6After incubating 10 HL-60 cells for 10 minutes, they were lysed for 10 minutes, 20 minutes, and 30 minutes respectively, and then the activities of glucose oxidase, lactate dehydrogenase, and alkaline phosphatase were measured. The detection wavelengths λ were 550 nm, 450 nm, and 520 nm respectively. Three aliquots of 10 6 HL-60 cells were lysed with traditional RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS) for 10 minutes, and then lysed for 10 minutes, 20 minutes, and 30 minutes respectively. Subsequently, the activities of glucose oxidase, lactate dehydrogenase, and alkaline phosphatase were measured, and the detection wavelengths λ were 550 nm, 450 nm, and 520 nm respectively. The results are shown in Figure 5 .

[0100] As can be seen from Figure 5 , after extraction with the ALTD cell lysis buffer of the present invention, the activities of the three enzymes are high. Moreover, as the treatment time increases, the decline in the activities of the three enzymes is relatively slow. In contrast, after extraction with the traditional RIPA lysis buffer, the activities of the three enzymes are low, and as the lysis time increases, the decline in the activities of the three enzymes is rapid. This indicates that the ALTD cell lysis buffer provides better protection for the activities of different enzymes in HL-60 cells and can ensure the stability of the structures of different enzymes, indirectly protecting the enzyme activities.

[0101] Comparative Example 3

[0102] After treating HL-60 cells with the cell lysis buffer in Example 1 according to the kit in Example 4 and the detection method in Example 5, the samples were diluted 1-fold, 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, and 64-fold, and the activities of glucose oxidase at 7 dilution multiples were measured with a blank added. The detection wavelength λ was 550 nm. After treating HL-60 cells with the traditional RIPA lysis buffer, the samples were diluted 1-fold, 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, and 64-fold, and the activities of glucose oxidase at 7 dilution multiples were measured with a blank added. The activities of glucose oxidase were measured using a commercially available kit (Company: Elibscience, Product Catalog Number Name: E-BC-K520-M; Glucose Oxidase (GOD) Colorimetric Assay Kit) according to the detection method described in its attached instruction manual. The results are shown in Table 2.

[0103] Table 2

[0104]

[0105] As can be seen from Table 2, when the ALTD cell lysis buffer of the present invention is combined with the kit, compared with the combination of RIPA and the commercially available kit, the signal-to-noise ratio window is larger: 65 vs 10. The sample dilution curve is as shown in Figure 6As shown, it has a wider linear range and better linearity. It has higher sensitivity, with a minimum concentration of 0.003 U / ml. When the cell lysate and kit of the present invention are used, it can be more clearly distinguished from the background 0 point.

[0106] It should be noted that only some of the numerous enzymes in the cell (such as glucose oxidase, lactate dehydrogenase, alkaline phosphatase) are used in the experiment of the present invention, but it should not be construed as a limitation on the protection scope of the present invention.

[0107] Compared with the prior art, the beneficial effects of the present invention include:

[0108] (1) Compared with the existing lysates, the lysate of the present invention can lyse cells through the combination of 1% Triton X-100 and 0.25% sodium deoxycholate to release the enzymes, while avoiding over-lysis and inactivation due to structural changes. The enzyme activity protectant and enzyme structure stabilizer mentioned above act synergistically to protect the enzyme. The former directly acts on the active site of the enzyme to play a protective role, and the latter acts on the enzyme composition structure to indirectly protect the enzyme activity. Among them, the combination of 0.1-0.5% β-cyclodextrin and 0.1-0.5% sodium carboxymethyl cellulose, the combination of 10-20 g / L mannitol and 0.1-0.5% dextran, and the combination of 0.5-1.5% PVP and 1-5% PEG6000 act synergistically. These three low-molecular and high-molecular substances act synergistically to protect different types of enzymes respectively, ensuring the stability of the enzyme structure.

[0109] (2) The present invention reduces the difficulty of sample processing, is easier to operate, and has a shorter detection time.

[0110] (3) The present invention uses a fixation technique to fix the enzyme or substrate and enzyme at the bottom of the enzyme-labeled plate. The reagents are reduced and the stability time is longer. Combined with the lysate of the present invention, it protects the enzyme to be detected in the cell sample and the fixed tool enzyme that plays a detection role in the enzyme-labeled plate. Compared with the ordinary lysate that will have an obvious impact on the enzyme activity and the tool enzyme in the detection system, the fixation technique combined with the lysate of the present invention can use a tool enzyme with higher enzyme activity to detect the enzyme to be detected with higher enzyme activity, and has higher sensitivity when detecting the same cell sample and the same enzyme.

[0111] (4) The method of treating with the lysate of the present invention, compared with the conventional ordinary mechanical homogenization, does not need to add steel beads, does not need to vibrate and grind on a homogenizer, and does not need to take out the steel beads after homogenization; compared with the ultrasonic fragmentation method, it does not need an ultrasonic crusher and does not need to perform single fragmentation treatment. The lysate of the present invention can process more samples in the same time, and does not require expensive instrument equipment, providing the possibility for meeting the high-throughput sample processing and detection needs of customers.

[0112] The specific embodiments of the present invention described above do not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and deformations made according to the technical concept of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A cell lysis solution, characterized in that: The components of the cell lysate include: 25-150 mM first buffer, 75-125 mM isotonic component, 1wt% Triton X-100, 0.25wt% sodium deoxycholate, 1-2 mM EDTA, 0.1-0.5wt% BSA, 20-80 g / L sucrose, 1-3wt% glycerol, 0.1-0.5wt% β-cyclodextrin, 10-20 g / L mannitol, 10-20 g / L hexose, 0.1-0.5wt% sodium methylcellulose, 10-25 mM sodium sulfate, 0.1-0.5wt% dextran, 0.5-1.5wt% PVP, 5-10mM glycine, and 1-5wt% PEG6000; wherein the first buffer is Tris-HCl with a pH of 7-8; and the isotonic component is NaCl.

2. A kit for detecting enzyme activity in cells, characterized in that: The cell enzyme activity detection kit comprises the cell lysate according to claim 1.

3. The cell enzyme activity detection kit according to claim 2, characterized in that: The cell enzyme activity detection kit is a cell enzyme activity detection kit based on fixation technology.

4. The cell enzyme activity detection kit according to claim 3, characterized in that: The cell enzyme activity detection kit based on the fixation technology also includes: a coated ELISA plate, an enzyme activity detection colorimetric agent, and optionally a standard substance.

5. Use of the cell enzyme activity detection kit as claimed in claim 2 in detecting the enzyme activity in cells.

6. Use of the cell enzyme activity detection kit according to claim 5 in detecting the enzyme activity in cells, characterized in that: The cell enzyme activity detection kit is a cell enzyme activity detection kit based on fixation technology, and the application includes the following steps: S1. Sample preparation: Collect 10 5 -10 6 Wash the cell samples to be tested thoroughly with the pre-cooled second buffer; S2. Sample processing: add 100-200 μL of the cell lysis solution to the collected cells, mix well and place on ice for lysis, centrifuge and take the supernatant to obtain the sample to be tested; S3. Standard preparation: dilute with cell lysate to obtain a series of standard solutions with different concentrations; S4, determination: set standard wells and determination wells on the coated ELISA plate, add V1 volume of a series of standard solutions of different concentrations to the standard wells, add V1 volume of the sample to be tested to the determination wells, and then add V2 volume of enzyme activity detection colorimetric reagent to the standard wells and the determination wells, mix well and incubate at temperature T, detect OD values ​​A1 and A2 at two fixed time points at a wavelength of λnm using an ELISA reader, and calculate the change value △A; S5. Draw a standard curve and calculate the enzyme activity using the universal cell enzyme activity formula shown in formula (1); wherein formula (1) is as follows: X enzyme activity (U / gprot) = (△Ab) ÷ a ÷ C pr ÷ t × f (1); In formula (1), a is the slope of the standard curve, b is the intercept of the standard curve, and t is the interval time min between two fixed time points; C pr is the protein content in the sample to be tested after centrifugation in step S2; f is the dilution multiple of the sample to be tested before adding it to the well of the ELISA plate after centrifugation in the sample processing step; the enzyme activity in formula (1) is defined as: at temperature T, the amount of enzyme that catalyzes the production of 1 μmol of product per gram of protein per minute is 1 activity unit; or the enzyme activity in formula (1) is defined as: at temperature T, the amount of enzyme that catalyzes the consumption of 1 μmol of substrate per gram of protein per minute is 1 activity unit.

7. Use of the cell enzyme activity detection kit according to claim 6 in detecting the enzyme activity in cells, characterized in that: In step S1, the second buffer is a phosphate buffer with a concentration of 0.005-0.02 M and a pH of 7-8.

8. Use of the cell enzyme activity detection kit according to claim 6 in detecting the enzyme activity in cells, characterized in that: In step S2, the pyrolysis temperature is 0-8°C, and the pyrolysis time is 5-40 min.

9. Use of the cell enzyme activity detection kit according to claim 6 in detecting the enzyme activity in cells, characterized in that: In step S4, V1 is 1-250 μL, V2 is 50-299 μL, temperature T is 32-40° C., and detection wavelength λ is 260-800 nm; in step S5, t is 0.5-120 min.

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