Method for detecting biological activity of GDF-11

Abstract

The invention provides a method for detecting the biological activity of GDF-11, which comprises the following steps: detecting the number of MPC-11 cells through a cell activity kit, analyzing the relationship between the dosage concentration of GDF-11 and the number of the MPC-11 cells, and calculating the half inhibitory concentration IC50, thereby evaluating the biological activity of GDF-11. The method has the advantages of large detection window, simple detection method, easily available raw materials, low cost and good result stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular, to a detection method of GDF-11 biological activity. BACKGROUND

[0002] For the detection of GDF-11 activity, there are currently two schemes as follows:

[0003] Scheme one: the detection of GDF-11 activity is realized by using osteoblasts MC3T3-E1 and chondrocytes ATDC5. However, the osteoblasts MC3T3-E1 and chondrocytes ATDC5 have the shortcomings of great difficulty in culture, easy differentiation and non-response to factors. Moreover, the detection process needs GDF-11 and bone morphogenetic protein 2 (BMP2) to be incubated with cells, and then the expression amount of alkaline phosphatase ALP is detected, which has the shortcomings of complicated operation steps (cell washing, lysis, supernatant extraction, and detection of ALP expression amount by pNPP or 4MUP substrate), certain failure rate, large operation error and low window.

[0004] Scheme two: the detection of GDF-11 activity is realized by using GDF-11 to induce K562 cells to express hemoglobin protein. However, the scheme has the shortcomings that the hemoglobin protein detection needs to lyse cells, and the operation of protein extraction is difficult. Moreover, if TMB-H2O2 color reagent is used, there is the problem of low window and unstable results; and if hemoglobin ELISA kit is used, there is the problem of high cost and inconvenience for large-scale detection.

[0005] Therefore, new ideas and strategies for GDF-11 activity detection are to be developed. SUMMARY

[0006] The present application aims to overcome the above-mentioned defects, and provides a detection method of GDF-11 biological activity with large detection window, simple detection method, easy-to-obtain raw materials and low cost.

[0007] The present application discloses the application of MPC-11 in the detection of GDF-11 biological activity.

[0008] The present application discloses the application of MPC-11 in the preparation of GDF-11 biological activity detection products.

[0009] In addition, the present application also provides a GDF-11 biological activity detection product, which comprises a cell viability detection kit and an MPC-11 preparation.

[0010] In addition, the application also provides a GDF-11 biological activity detection method, which detects the MPC-11 cell quantity through a cell viability kit, analyzes the relationship between the GDF-11 dose concentration and the MPC-11 cell quantity, and calculates the half-inhibitory concentration IC50, so as to evaluate the biological activity of GDF-11.

[0011] Effects of the application:

[0012] The MPC-11 used in the application is a suspension cell, the culture medium has simple components and no differentiation risk, and the culture difficulty is low. The detection step only needs to add a cell viability kit for one-step reaction, and then multifunctional enzyme labeling instrument can be used to record data for analysis, the detection window is larger, the result is more stable, and the step is simple and convenient to operate. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 , the result graph of the detection according to the method of embodiment 1.

[0014] Figure 2 , the comparison graph of the two detection methods.

[0015] Figure 3 , the result graph of the detection according to the method of ATDC5. DETAILED DESCRIPTION

[0016] The application can be implemented in various ways and can have various embodiments, so specific embodiments are illustrated in the drawings and described. However, this is not intended to limit the application to specific embodiments, but should be understood to include all modifications, equivalents and even alternatives falling within the spirit and technical scope of the application.

[0017] Embodiment 1. GDF-11 biological activity detection method

[0018] Through the research of the application, it is found that GDF-11 can inhibit MPC-11, so in this embodiment, the characteristics of GDF-11 regulating MPC-11 are used, the MPC-11 cell quantity is detected through a cell viability kit, the relationship between the GDF-11 dose concentration and the MPC-11 cell quantity is analyzed, and the half-inhibitory concentration (IC50) is calculated, so as to evaluate the biological activity of GDF-11 (the smaller the IC50 value, the better the activity).

[0019] The detection principle of the cell viability kit is that the total amount of ATP is determined by the number of cells in the detection hole, the detection reagent reacts with ATP in the detected cells to emit special fluorescence, and the intensity of light is linearly related to the number of cells in a certain range through a multifunctional enzyme labeling instrument.

[0020] Experimental materials:

[0021]

[0022] The specific detection steps are as follows:

[0023] S0. Cell viability detection kit preparation: Take one bottle of substrate freeze-dried powder and one bottle of buffer (100 ml / bottle) from -80°C and equilibrate to room temperature. Transfer 100 ml of buffer to a brown bottle containing the substrate freeze-dried powder, mix gently by vortexing, rotating or inverting the contents up and down, and aliquot 10 ml / bottle after complete dissolution, and transfer to -80°C for storage. After reconstitution, the CTG Reagent can be stored at room temperature for up to 8 hours.

[0024] S1. Culture MPC-11 cells in MPC-11 cell-specific medium. Mix 20 μl of cell suspension with 20 μl of trypan blue reagent, and transfer 20 μl of the mixture to a cell counting plate. Use a cell counter to detect cell density and viability. The cell density should be between 0.05-0.3 x 106 cells / ml, and the viability should be greater than 90%.

[0025] S2. Wash the cells twice with PBS, resuspend the cells in MPC-11 cell-specific medium to the appropriate density, and inoculate the cells into Assay Plate wells B2 to C10 (2 rows x 9 columns). Inoculate 50 μl of cells per well (about 3000 cells per well).

[0026] S3. Dilute the GDF11 sample in MPC-11 cell-specific medium to concentrations of 4000, 2000, 500, 125, 31.25, 7.813, 1.953, 0.488 ng / ml, and use Cell only and medium as blank groups.

[0027]

[0028] S4. Transfer 50 μl of the gradient-diluted protein to the corresponding experimental wells of the Assay Plate. Set up two replicate wells for each sample concentration (or three replicate wells to improve the reliability of experimental data). Add 100 μl of PBS to the peripheral wells.

[0029] Example of experimental well plate:

[0030]

[0031] S5. Transfer the Assay Plate to a carbon dioxide incubator for incubation for 72 hours.

[0032] S6. Take out the well plate, shake it at 700 rpm for 3 min, and equilibrate it at room temperature for 30 min.

[0033] S7. Take out the cell viability detection reagent and equilibrate it to room temperature. Add 50 ul / well to the well plate.

[0034] S8. Mix the orifice plate by shaking at 700 rpm for 3 min, and let it stand at room temperature for 10 min.

[0035] S9. Take 80 μL of the reaction mixture and transfer it to a 96-well white microplate. Record the chemiluminescence value using a multi-functional microplate reader.

[0036] S10. Raw data from the cell proliferation experiment were exported and analyzed using Microsoft Office Excel and GraphPad Prism 8 software. The data were imported into GraphPad Prism 8 software with the final concentration of each sample in the reaction as the X-axis and the relative chemiluminescence unit as the Y-axis. A dose-response curve was established using a four-parameter equation to obtain the best fit value and goodness-of-fit data. An example of data processing is shown below. Figure 1 As shown.

[0037] Example 2. Comparative Experiment

[0038] A. Detection Window

[0039] like Figure 2 As shown in the figure, the results of testing the same sample using the method of this embodiment (left) and the chondrocyte ATDC5 method (right) can be seen from the figure.

[0040] In this embodiment, the detection window = Top / Bottom = 11641937 / 1107762 = 10;

[0041] In the ATDC5 assay for chondrocytes, the detection window = Top / Bottom = 1336 / 788.1 = 1.69;

[0042] In other words, the detection window of the method in this embodiment is much larger than the detection window of the ATDC5 method.

[0043] B. Accuracy

[0044] Using the same sample from Example 1, the data obtained by testing with MPC-11 using the method of Example 1 were stable, with small differences between replicates and more accurate IC50 values.

[0045] However, when using ATDC5 for detection, the differences between duplicate wells are significant, and the fitted IC50 is greatly affected by the fluctuations between duplicate wells. Figure 3 The image shows the results of parallel testing. From this result and... Figure 2 The results (right) show that the results of the same sample tested twice are completely different. In other words, the ATDC5 method has unstable results and requires multiple measurements to determine the average IC50 value.

[0046] While the above has been described in detail with respect to the embodiments, this is only illustrative and not restrictive. It will be obvious to those skilled in the art that various changes and modifications can be made without departing from the essence of the embodiments. For example, various elements of the embodiments can be altered or replaced by other elements. In addition, various differences in the embodiments related to such alterations and modifications should be interpreted as falling within the scope of the application as defined in the appended claims.

Claims

1. Use of MPC-11 in detection of biological activity of GDF-11.

2. Use of MPC-11 in preparation of detection product of biological activity of GDF-11.

3. A product for detecting the biological activity of GDF-11, characterized by: A cell viability detection kit and a preparation of MPC-11.

4. A method for detecting the biological activity of GDF-11, characterized by: The number of MPC-11 cells is detected by the cell viability kit, the relationship between the dose concentration of GDF-11 and the number of MPC-11 cells is analyzed, and the half-inhibitory concentration IC50 is calculated, so as to evaluate the biological activity of GDF-11.

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