Method for detecting activity of glucocerebrosidase drugs based on cell intake

Abstract

The invention discloses a method for detecting the activity of glucocerebrosidase drugs based on cell intake, and the method comprises the following steps: (a) enabling cells to intake glucocerebrosidase drugs, and enabling the cells to overexpress mannose receptors; (b) removing the drug which is non-specifically bound outside the cells; (c) lysing the cells; and (d) carrying out enzyme specific activity detection on the lysate.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, specifically relates to the field of activity detection, and particularly relates to a cell-based bioactivity detection method for detecting glucocerebrosidase drugs by using CHO (Chinese Hamster Ovary) cells overexpressing mannose receptor. Background Art

[0002] Gaucher disease is an autosomal recessive genetic disease caused by gene mutations, resulting in the deficiency of glucocerebrosidase (GCase) and the accumulation of its substrate glucocerebroside in the lysosomes of macrophages. The clinical manifestations of this disease include hepatosplenomegaly, anemia, bone pain, neurological symptoms, etc.

[0003] Currently, enzyme replacement therapy (ERT) is the main treatment method for this disease. By intravenous infusion (about once every two weeks), the recombinant form of the enzyme is used to compensate for the lack of GCase activity in patients with Gaucher disease. Approved drug examples include imiglucerase, velaglucerase alfa, and taliglucerase alfa. Glucocerebrosidase has mannose residues and can enter cells by binding to mannose receptors on the cell surface and then enter lysosomes to effectively hydrolyze glucocerebroside.

[0004] Quantitative detection and analysis of the biological activity of enzyme drugs according to their characteristic properties is a prerequisite for ensuring the safety and effectiveness of drugs. For the activity evaluation of glucocerebrosidase drugs, there are biochemical methods and cell-based detection methods. Traditional biochemical methods include colorimetry, fluorescence method, and enzyme-linked immunosorbent assay. The detection principle is based on the hydrolysis of substrates (such as p-nitrophenyl-β-D-glucopyranoside and 4-methylumbelliferyl-β-D-glucopyranoside) by glucocerebrosidase to produce products with fluorescent properties, and the enzyme activity is evaluated by quantitative detection. The bioactivity pharmacodynamic evaluation based on cell methods can efficiently, rapidly, and directly establish an analysis method with biological relevance and can reflect the biological activity of drugs from a perspective closer to the in vivo mechanism.

[0005] However, if a general natural cell line is used for the intake and bioactivity analysis of glucocerebrosidase drugs, there will be a problem of non-specific binding between the enzyme drugs and the cells, resulting in inaccurate activity evaluation. The intake of glucocerebrosidase drugs by natural macrophages continuously increases over time, and it is impossible to perform four-parameter fitting to generate a complete curve, which is not conducive to the evaluation of the relative activity of the drugs. Solving the problem of non-specific binding can saturate the intake of enzyme drugs within a certain time, and then the activity titer of the drugs can be evaluated more accurately.

[0006] There is still an urgent need in the art for a cell-based detection method that can avoid the interference of non-specific binding of drugs, eliminate the influence of continuous intake of glucocerebrosidase drugs, and thus improve the evaluation effect of the relative activity of drugs. Summary of the Invention

[0007] To solve the above technical problems, the present application provides a method for detecting the activity of glucocerebrosidase drugs based on cell intake, and the method includes:

[0008] (a) Allowing cells to intake glucocerebrosidase drugs, wherein the cells overexpress mannose receptors;

[0009] (b) Removing the drugs non-specifically bound outside the cells;

[0010] (c) Lysing the cells; and

[0011] (d) Detecting the specific activity of the enzyme in the lysate. Brief Description of the Drawings

[0012] The present application will be described in more detail below with reference to the accompanying drawings, in which:

[0013] Figure 1 is the standard curve of the enzyme activity reaction.

[0014] Figure 2 is the standard curve for protein concentration detection.

[0015] Figure 3 is the specific activity result of the enzyme drug. Detailed Description of the Embodiments

[0016] This application relates to a method for detecting the activity of glucocerebrosidase drugs based on cell uptake, which method comprises: (a) causing a cell that overexpresses a mannose receptor to take up a glucocerebrosidase drug. In one embodiment, the glucocerebrosidase drug comprises glucocerebrosidase. In one embodiment, the glucocerebrosidase drug comprises β-glucocerebrosidase. In one embodiment, the cell overexpresses the mannose receptor on the cell surface. In one embodiment, the cell comprises a mammalian cell. In one embodiment, the cell comprises a macrophage. In a preferred embodiment, the cell comprises a CHO cell. In a preferred embodiment, the cell comprises a CHO cell that overexpresses the mannose receptor. In one embodiment, the overexpression is achieved by engineering. In one embodiment, the overexpression comprises: the cell does not express the mannose receptor before engineering, and the cell expresses the mannose receptor after engineering. In one embodiment, the overexpression comprises that the amount of the mannose receptor expressed by the cell after engineering is increased by at least 20%. In one embodiment, the overexpression comprises that the amount of the mannose receptor expressed by the cell after engineering is increased by at least 50%. In one embodiment, the overexpression comprises that the amount of the mannose receptor expressed by the cell after engineering is increased by at least 100%. In one embodiment, the overexpression comprises that the amount of the mannose receptor expressed by the cell after engineering is increased by at least 200%. In one embodiment, step (a) comprises contacting the cell with the glucocerebrosidase drug. In one embodiment, step (a) is carried out in a multi-well plate. In a preferred embodiment, step (a) is carried out in a 96-well plate. In one embodiment, the method further comprises spreading a cell suspension on the multi-well plate and incubating it before step (a). In one embodiment, the cell suspension in the multi-well plate is 100 - 200 μL / well. In one embodiment, the cell suspension in the multi-well plate is about 150 μL / well. In one embodiment, the incubation lasts no more than 5 hours. In one embodiment, the incubation lasts no more than 2 hours. In one embodiment, the incubation lasts no more than 1 hour. In a preferred embodiment, the incubation lasts about 0.5 hour. In one embodiment, the cell density in the multi-well plate is 1×10 5 to 1×10 7 cells / well. In one embodiment, the cell density in the multi-well plate is 5×10 5 to 1×10 6 cells / well. In a preferred embodiment, the cell density in the multi-well plate is about 9×10 5cells / well. In one embodiment, the incubation temperature is 35 - 40 °C. In one embodiment, the incubation temperature is 36 - 38 °C. In a preferred embodiment, the incubation temperature is about 37 °C. In one embodiment, the incubation conditions include carbon dioxide. In a preferred embodiment, the incubation conditions include about 5% carbon dioxide. In one embodiment, the method further comprises diluting the glucocerebrosidase drug before step (a). In one embodiment, the dilution includes serial dilution. In one embodiment, the amount of the glucocerebrosidase drug added is 10 - 100 μl / well. In one embodiment, the amount of the glucocerebrosidase drug added is 30 - 70 μl / well. In a preferred embodiment, the amount of the glucocerebrosidase drug added is about 50 μl / well. In one embodiment, the final concentration of the glucocerebrosidase drug is 50 ng / ml - 300 μg / ml. In a preferred embodiment, the final concentration of the glucocerebrosidase drug is 128.8 ng / ml - 175.0 μg / ml. In one embodiment, the contact includes attaching a respiratory membrane after adding the glucocerebrosidase drug and then performing the contact after shaking. In one embodiment, the shaking lasts for 1 - 30 minutes. In a preferred embodiment, the shaking lasts for 5 - 10 minutes. In one embodiment, the shaking is carried out on a shaker. In one embodiment, the contact lasts for 4 - 36 hours. In one embodiment, the contact lasts for 5 - 30 hours. In a preferred embodiment, the contact lasts for 15 - 19 hours. In a preferred embodiment, the contact lasts for about 17 hours. In one embodiment, the contact temperature is 35 - 40 °C. In one embodiment, the contact temperature is 36 - 38 °C. In one embodiment, the contact temperature is about 37 °C. In one embodiment, the contact conditions include carbon dioxide. In a preferred embodiment, the contact conditions include about 5% carbon dioxide.

[0017] The method for detecting the activity of glucocerebrosidase drugs further includes: (b) removing the drugs non-specifically bound outside the cells. In one embodiment, step (b) includes treating the cells with trypsin, terminating with a digestion termination solution, and washing the cells. In one embodiment, the washing includes washing the cells with a buffer solution. In one embodiment, the washing includes washing the cells with PBS. In one embodiment, step (b) includes removing the liquid in the plate before treating the cells with trypsin. In one embodiment, trypsin is added as a solution. In one embodiment, the concentration of the trypsin solution is 0.1%-0.5%. In one embodiment, the concentration of the trypsin solution is 0.2%-0.3%. In a preferred embodiment, the concentration of the trypsin solution is about 0.25%. In one embodiment, the addition amount of the trypsin solution is 10-100 μl / well. In one embodiment, the addition amount of the trypsin solution is 30-70 μl / well. In a preferred embodiment, the addition amount of the trypsin solution is about 50 μl / well. In one embodiment, treating the cells with trypsin includes shaking and then incubating. In one embodiment, the shaking is carried out at room temperature. In one embodiment, the shaking lasts for 1-10 minutes. In a preferred embodiment, the shaking lasts for about 5 minutes. In one embodiment, the incubation temperature is 35-40 °C. In one embodiment, the incubation temperature is 36-38 °C. In a preferred embodiment, the incubation temperature is about 37 °C. In one embodiment, the incubation conditions include carbon dioxide. In a preferred embodiment, the incubation conditions include about 5% carbon dioxide. In one embodiment, the incubation lasts for 1-30 minutes. In a preferred embodiment, the incubation lasts for about 5-10 minutes. In one embodiment, the digestion termination solution includes a buffer solution containing HI-FBS. In one embodiment, the digestion termination solution includes DPBS containing HI-FBS. In a preferred embodiment, the digestion termination solution includes DPBS containing about 10% HI-FBS. In one embodiment, the addition amount of the digestion termination solution is 20-200 μl / well. In one embodiment, the addition amount of the digestion termination solution is 50-150 μl / well. In a preferred embodiment, the addition amount of the digestion termination solution is about 100 μl / well. In one embodiment, the washing includes removing the liquid in the plate. In one embodiment, the washing further includes, after removing the liquid in the plate, washing the plate with pre-cooled DPBS and resuspending the cells. In one embodiment, the addition amount of the pre-cooled DPBS is 100-500 μl / well. In one embodiment, the addition amount of the pre-cooled DPBS is 200-300 μl / well. In a preferred embodiment, the addition amount of the pre-cooled DPBS is about 250 μl / well. In one embodiment, the washing is repeated multiple times. In a preferred embodiment, the washing is repeated three times.

[0018] The method for detecting the activity of glucocerebrosidase drugs further includes: (c) lysing the cells. In one embodiment, step (c) may include any technical means for lysing cells, such as lysing agents, sonication, homogenization, ball milling, heat denaturation, acid-base treatment, freeze-thaw, etc. In one embodiment, step (c) includes adding a lysing agent. In one embodiment, the lysing agent includes a mammalian protein extractant. In one embodiment, the lysing agent includes a protease inhibitor. In a preferred embodiment, the lysing agent includes a mammalian protein extractant containing a protease inhibitor. In one embodiment, the addition amount of the lysing agent is 10 - 500 μl / well. In one embodiment, the addition amount of the lysing agent is 50 - 200 μl / well. In a preferred embodiment, the addition amount of the lysing agent is about 100 μl / well. In one embodiment, the lysis temperature is 35 - 40 °C. In one embodiment, the lysis temperature is 36 - 38 °C. In a preferred embodiment, the lysis temperature is about 37 °C. In one embodiment, the lysis conditions include carbon dioxide. In a preferred embodiment, the lysis conditions include about 5% carbon dioxide. In one embodiment, the lysis lasts for 30 - 120 minutes. In a preferred embodiment, the lysis lasts for about 60 minutes.

[0019] The method for detecting the activity of glucocerebrosidase drugs further includes: (d) detecting the specific activity of the lysate. In one embodiment, step (d) includes reacting the lysate with a substrate. In one embodiment, the reaction lasts for 30 minutes to 2 hours. In a preferred embodiment, the reaction lasts for about 1 hour. In one embodiment, the reaction is carried out in an enzyme activity reaction plate. In one embodiment, the substrate includes p-nitrophenyl-β-D-glucopyranoside or 4-methylumbelliferyl-β-glucopyranoside (4-MU-β-Glucoside). In one embodiment, the substrate includes 4-methylumbelliferyl-β-glucopyranoside. In one embodiment, the substrate is added in solution. In one embodiment, the concentration of the substrate solution is 1-20 mM. In one embodiment, the concentration of the substrate solution is 2-10 mM. In a preferred embodiment, the concentration of the substrate solution is about 5 mM. In one embodiment, the added volume of the substrate solution is the same as the volume of the lysate in the well. In one embodiment, the added amount of the substrate solution is 10-50 μl / well. In one embodiment, the added amount of the substrate solution is 20-30 μl / well. In a preferred embodiment, the added amount of the substrate solution is about 25 μl / well. In one embodiment, the specific activity detection includes making a product standard curve. In one embodiment, the product may include any substance produced by the catalysis of the substrate by glucocerebrosidase drugs. In one embodiment, the product includes 4-MU (4-methylumbelliferone). In one embodiment, making the product standard curve includes dilution, such as serial dilution. In one embodiment, the dilution range is 0.01-20 nmol / well. In a preferred embodiment, the dilution range is 0.02-8.00 nmol / well. In one embodiment, the dilution concentrations include 8.00 nmol / well, 2.86 nmol / well, 1.02 nmol / well, 0.36 nmol / well, 0.13 nmol / well, 0.05 nmol / well, and 0.02 nmol / well. In one embodiment, 4-MU is diluted with the enzyme activity detection solution. In one embodiment, the enzyme activity detection solution includes NaOAc, NaCl, BSA, Triton-X100, with a pH of 4-5.5. In a preferred embodiment, the enzyme activity detection solution includes 50 mM NaOAc, 130 mM NaCl, 0.1 mg / mL BSA, 0.2% Triton-X100, pH 4.8. In one embodiment, step (d) further includes terminating the reaction with a termination solution. In a preferred embodiment, the termination solution includes glycine. In one embodiment, the concentration of glycine is 0.5-2 M. In one embodiment, the concentration of glycine is 0.8-1.2 M. In a preferred embodiment, the concentration of glycine is about 1 M. In one embodiment, the added amount of the termination solution is 50-200 μl / well.In one embodiment, the addition amount of the termination solution is 100 - 150 μl / well. In a preferred embodiment, the addition amount of the termination solution is about 125 μl / well. In one embodiment, step (d) further includes transferring the reaction system to an enzyme activity detection plate after adding the termination solution. In one embodiment, the amount of the transferred reaction system is 50 - 200 μl. In one embodiment, the amount of the transferred reaction system is 100 - 150 μl. In a preferred embodiment, the amount of the transferred reaction system is about 130 μl. In one embodiment, step (d) further includes reading the fluorescence value after the transfer. In one embodiment, step (d) further includes reading the fluorescence value after a period of time. In one embodiment, the fluorescence value includes relative fluorescence units (RFU). In one embodiment, the period of time includes 20 - 40 minutes. In a preferred embodiment, the period of time includes about 30 minutes. In one embodiment, step (d) further includes reading the fluorescence value with a microplate reader. In one embodiment, step (d) includes measuring the enzyme activity value, the wherein the actually measured amount of 4-MU generated per well is calculated by the 4-MU standard curve, and the amount of 4-MU produced in each sample well is calculated using the RFU value measured in the well where the sample is located. In one embodiment, the 4-MU standard curve is generated by four-parameter fitting. In one embodiment, the formula for four-parameter fitting is as follows: where y represents the fluorescence value, B represents the slope, and C represents EC 50Half maximal effective concentration, where x represents concentration, A represents the lower plateau, and D represents the upper plateau. In one embodiment, y includes relative fluorescence units (RFU). In one embodiment, the four-parameter fitting is performed and calculated by softmax software. In one embodiment, step (d) further includes protein concentration determination. In one embodiment, the protein concentration determination includes the BCA protein quantification method. In one embodiment, the BCA protein quantification method includes diluting the cell lysate, adding the BCA working solution, incubating, and reading the absorbance value with an enzyme-linked immunosorbent assay (ELISA) reader. In a preferred embodiment, the BCA protein quantification method includes diluting the cell lysate 4.8-fold with ultrapure water, adding an equal volume of the BCA working solution, incubating for 1.5 hours, and reading the absorbance value with an ELISA reader within 30 minutes. In one embodiment, the absorbance value includes the absorbance value at a wavelength of 550 - 580 nm. In one embodiment, the absorbance value includes the absorbance value at a wavelength of 560 - 565 nm. In a preferred embodiment, the absorbance value includes the absorbance value at a wavelength of approximately 562 nm. In one embodiment, the protein concentration determination includes preparing a reference standard curve. In one embodiment, the reference includes BSA (bovine serum albumin). In one embodiment, preparing the product standard curve includes dilution, such as serial dilution. In one embodiment, the dilution range is from 0.01 - 1 mg / mL. In one embodiment, the dilution range is from 0.021 - 0.667 mg / mL. In one embodiment, the dilution concentrations include 0.667 mg / mL, 0.333 mg / mL, 0.167 mg / mL, 0.083 mg / mL, 0.042 mg / mL, and 0.021 mg / mL. In one embodiment, BSA is diluted with the lysate. In one embodiment, the lysate contains protease inhibitors. In one embodiment, the In a preferred embodiment, the protein concentration is calculated from the bovine serum albumin (BSA) standard curve generated by four-parameter fitting. In one embodiment, the formula for four-parameter fitting is as follows: where y represents the absorbance value (OD), B represents the slope, and C represents the EC 50 Half maximal effective concentration, where x represents concentration, A represents the lower plateau, and D represents the upper plateau. In one embodiment, y includes the absorbance value at a wavelength of 550 - 580 nm. In one embodiment, y includes the absorbance value at a wavelength of 560 - 565 nm. In a preferred embodiment, y includes the absorbance value at a wavelength of approximately 562 nm. In one embodiment, the four-parameter fitting is performed and calculated by softmax software.

[0020] Examples

[0021] This application will be described in detail through the following exemplary specific embodiments. The following embodiments are only used to help those skilled in the art better understand various inventions of this application. It should be pointed out that the spirit of this application and the protection scope of the claims are not limited by the following specific embodiments.

[0022] List of reagents and equipment used

[0023]

[0024]

[0025] In the following embodiments, if the reagents used are not specifically stated, they are commercial products.

[0026] 1) Suspend the suspension of engineered overexpressing mannose receptor CHO cells (produced by WuXi-Bio) at a density of 9×10 5 cells / well and a volume of 150 μl / well into a 96-well cell culture plate, and incubate it for 32 minutes at 37°C under 5% carbon dioxide conditions.

[0027] 2) Gradient dilute the samples (produced by WuXi-Bio), quality control products (produced by WuXi-Bio), and standard products (produced by WuXi-Bio) of glucocerebrosidase drugs in a 96-well dilution plate, with a concentration range of 515.1 ng / ml to 700.0 μg / ml.

[0028] 3) Add the drug to the cell culture well plate at a volume of 50 μL / well, then attach a breathing membrane and place it on a microplate shaker to shake the plate for 8 minutes. The final drug concentration range is 128.8 ng / ml to 175.0 μg / ml.

[0029] 4) Incubate the shaken cell culture plate at 37°C under 5% carbon dioxide conditions for 17 hours.

[0030] 5) Centrifuge the cell culture plate at 400 g and deceleration rate 1 (centrifuge eppendorf 5810R) for 5 minutes, and after centrifugation, discard the liquid in the plate and invert it on absorbent paper to suck out the liquid in the plate.

[0031] 6) Add 0.25% trypsin to the cell culture plate at a volume of 50 μl / well, gently shake it at room temperature for 5 minutes, and then transfer the cell culture plate to an incubator at 37°C under 5% carbon dioxide conditions and incubate for 10 minutes.

[0032] 7) After incubation, add the digestion termination solution (DPBS buffer containing 10% HI-FBS) to the cell culture plate at a volume of 100 μl / well and gently tap the well plate to mix evenly.

[0033] 8) After mixing each well with a multi-channel pipette, transfer all the cells to the corresponding positions on a 96-well V-bottom plate.

[0034] 9) Centrifuge the V-bottom plate at 400 g with a deceleration rate of 1 for 5 minutes at 2 - 8 °C. After discarding the liquid in the plate, wash the wells with pre-cooled (2 - 8 °C) DPBS at a volume of 250 μl / well, and pipette up and down to resuspend the cells at the bottom. Repeat the above washing process twice. After the last centrifugation, discard the liquid and invert the plate on absorbent paper to suck out the residual liquid.

[0035] 11) Add lysis buffer (mammalian protein extraction reagent containing Halt protease inhibitor cocktail) to the V-bottom plate at a volume of 100 μL / well. Transfer the plate to an incubator at 37 °C with 5% carbon dioxide for 63 minutes, and then mix the lysed cell lysate with a multi-channel pipette. TM 12) Dilute 4-MU with enzyme activity detection solution (50 mM NaOAc, (pH 4.8), 130 mM NaCl, 0.1 mg / mL BSA, 0.2% Triton-X100) to prepare 4-MU standard curve samples, with the dilution range of 0.02 - 8.00 nmol / well. The specific concentrations are shown in Table 1.

[0036] 13) Add the above lysed cell lysate to the 96-well enzyme activity incubation plate at a volume of 25 μl / well, and add 4-MU standard curve samples and blank control group (enzyme activity detection solution) to the plate at a volume of 50 μL / well.

[0037] Table 1. 4-MU standard curve concentrations

[0038]

[0039] 14) Add 5 mM 4-MU-β-glycoside working solution to all wells except for the 4-MU standard curve and blank control group (enzyme activity detection solution) at a volume of 25 μl / well.

[0040] 15) Seal the enzyme activity incubation plate with a sealing film and incubate it in an incubator at 37 °C with 5% carbon dioxide for 60 minutes for the enzyme activity reaction.

[0041] 16) Add 1 M glycine solution to all wells of the enzyme activity incubation plate at a volume of 125 μl / well, mix well, transfer to a 96-well enzyme activity detection plate, and read the relative fluorescence unit (RFU) value with an enzyme-linked immunosorbent assay (ELISA) reader (M5e from Molecular Devices, USA). First, generate a 4-MU standard curve by four-parameter fitting: plot the difference between the relative fluorescence unit values of different concentration points of the 4-MU solution and the blank control value on the Y-axis, and the logarithm of the concentration on the X-axis (nmol / well). Fit through the four-parameter equation (the 4-MU standard curve details are shown in

[0042] 17) Figure 1)。The four-parameter fitting is performed and calculated by softmax software. The specific formula for the four-parameter fitting is as follows:

[0043]

[0044] Among them, y represents relative fluorescence unit (RFU), B represents the slope, C represents EC 50 half maximal effective concentration, x represents the concentration, A represents the lower plateau, D represents the upper plateau, where A, B, C, and D are values obtained by fitting and calculation with softmax software. Then, through the 4-MU standard curve, the amount of 4-MU produced in each standard and sample well is calculated using the RFU values measured in the wells of the standard and sample. Then, the enzyme activity value of each well is calculated through the following formula:

[0045]

[0046] At the same time, the protein concentration is measured: A standard curve is prepared by diluting BSA with a lysis buffer containing protease inhibitors (without cells and contents), and the dilution range is 0.021 - 0.667 mg / mL (specific concentrations are shown in Table 2); the BSA stock solution in the protein concentration detection kit is diluted with a lysis buffer containing protease inhibitors to prepare a BSA standard product with a final concentration of 0.1 mg / mL.

[0047] Table 2. Concentrations of the BSA standard curve

[0048] Concentration mg / mL Example of detection plate reading OD value BSA-01 0.667 0.855 BSA-02 0.333 0.496 BSA-03 0.167 0.324 BSA-04 0.083 0.235 BSA-05 0.042 0.185 BSA-06 0.021 0.159

[0049] 17) Add BSA standard curve samples, BSA standard products, and lysed cell solutions to the 96-well protein detection plate at a volume of 25 μl / well. Add 95 μl of ultrapure water and 120 μL of BCA working solution to each well.

[0050] 18) Place the protein detection plate in an incubator at 37 °C and 5% carbon dioxide for 1 hour and 30 minutes, and then read the absorbance value (OD) at 562 nm using an enzyme-linked immunosorbent assay reader. First, generate a BSA standard curve using the four-parameter fitting method: Plot the average absorbance value (y-axis) of each concentration point of the BSA standard solution against the logarithmic concentration (x-axis), and perform four-parameter equation fitting using softmax software. Then, through the fitted BSA standard curve, calculate the protein concentration of the lysed cell solution using the absorbance value measured in the sample well ( Figure 2 ), and the four-parameter fitting formula is as follows:

[0051]

[0052] Among them, y represents the absorbance value (OD), B represents the slope, C represents EC 50Half maximal effective concentration, where x represents concentration, A represents the lower plateau, D represents the upper plateau, and A, B, C, and D are values calculated by fitting with softmax software.

[0053] 19) According to the obtained enzyme activity value and protein concentration value, calculate the specific enzyme activity value through the following formula:

[0054]

[0055] Then, with the specific enzyme activity value of the sample as the y-axis and the logarithm of the concentration as the x-axis, use softmax software to fit the four-parameter equation to generate the effect concentration curve (as Figure 3 ), and the four-parameter fitting formula is as follows:

[0056]

[0057] Among them, y represents the specific enzyme activity value, B represents the slope, C represents EC 50 Half maximal effective concentration, where x represents concentration, A represents the lower plateau, and D represents the upper plateau.

[0058] In summary, compared with the prior art, the present invention has the following advantages:

[0059] 1. The step of removing non-specific binding involved in the method of the present invention has broad application value for the activity evaluation of enzyme drugs by using cell uptake methods.

[0060] 2. The detection method provided by the present invention is conducive to the research and development, quality control, and clinical application of glucocerebrosidase drugs, and has high application value.

[0061] 3. Compared with the prior art, the method of the present invention can remove the products of non-specific binding on cells and the interference of drugs that are not taken into cells, and can accurately reflect the activity of enzyme drugs taken into cells, so as to accurately reflect the mechanism and potency of enzyme drugs after being taken into cells in in vitro experiments.

[0062] 4. The method of the present invention can fit a complete activity curve through four parameters, including the upper and lower plateaus, making the activity evaluation result more objective and reliable.

Claims

1. A method for detecting the activity of glucocerebrosidase drugs based on cell uptake, the method comprising: (a) allowing a cell overexpressing a mannose receptor to take up a glucocerebrosidase drug; (b) removing the drug non-specifically bound outside the cell; (c) lysing the cell; and (d) detecting the specific enzyme activity of the lysate.

2. The method according to claim 1, wherein the cell comprises a mammalian cell, preferably a CHO cell.

3. The method according to claim 1, wherein step (a) comprises contacting the cell with a glucocerebrosidase drug, preferably incubating, for example, for 4 - 36 hours, for example, for 5 - 30 hours, preferably for 15 - 19 hours.

4. The method according to claim 1, wherein step (b) comprises treating the cell with trypsin, terminating with a digestion termination solution, and washing the cell, preferably washing the cell with PBS.

5. The method according to claim 1, wherein step (c) comprises adding a lysing agent, preferably, the lysing agent comprises a protease inhibitor.

6. The method according to claim 1, wherein step (d) comprises reacting the lysate with a substrate.

7. The method according to claim 6, wherein the substrate comprises 4-MU-β-glycoside.

8. The method according to claim 6, wherein step (d) further comprises terminating the reaction with a termination solution, preferably glycine, and / or step (d) further comprises protein concentration determination.

9. The method according to claim 1, wherein step (d) comprises measuring the enzyme activity value, the wherein the actually measured amount of 4-MU generated per well is calculated by a 4-MU standard curve, and the amount of 4-MU produced in each sample well is calculated using the RFU value measured in the well where the sample is located; Preferably, the 4-MU standard curve is generated by four-parameter fitting, preferably, the formula for the four-parameter fitting is as follows: Among them, y represents the fluorescence value, B represents the slope, and C represents the EC 50 Half maximal effective concentration, x represents the concentration, A represents the lower plateau, and D represents the upper plateau.

10. The method according to claim 9, wherein step (d) further comprises measuring the specific enzyme activity value, and wherein preferably, the protein concentration is calculated by a bovine serum albumin (BSA) standard curve generated by four-parameter fitting. Preferably, the formula of the four-parameter logarithm is as follows: Among them, y represents the absorbance value (OD), B represents the slope, and C represents EC 50 The median effective concentration, x represents the concentration, A represents the lower plateau, and D represents the upper plateau.

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