Method for detecting titer of urelirelin
By using 96-well plates and Tris buffer to prepare standard solutions for urokinase potency assay, combined with microplate reader readings, the problems of large errors and low efficiency in existing methods are solved, achieving efficient and accurate detection results.
Patent Information
- Application Number
- CN202510860646.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-25
- Publication Date
- 2026-02-03
AI Technical Summary
Existing methods for detecting the potency of urokinase have problems such as large analytical errors, low detection efficiency, results that depend on environmental conditions, and cumbersome operation.
The reaction was carried out using 96-well plates. Standard solutions and test solutions were prepared using Tris buffer. The results were read by an ELISA reader, and a standard curve was plotted to reduce operational steps and environmental influences, thereby improving detection accuracy and efficiency.
This significantly improves the accuracy and efficiency of ursodeoxylin potency testing, stabilizes absorbance readings, reduces instrument and operational errors, and shortens testing time to 1 hour per plate.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and more specifically to a method for detecting the potency of urokinase. Background Technology
[0002] Urinary kallindinogenase, also known as human urinary kininogenase, is a tissue-type kininogenase extracted from fresh human urine. It is a glycoprotein composed of 238 amino acids that converts kininogen into kinin and kallidin, selectively dilating microvessels in ischemic brain tissue, improving local cerebral blood flow, and reducing the ischemic penumbra. Urinary kallindinogenase also has neuroprotective effects, inhibiting neuronal apoptosis and promoting the proliferation, migration, and differentiation of neural stem cells into mature neurons after cerebral ischemia, thereby playing a role in nerve repair. Furthermore, urinary kallindinogenase enhances fibrinolytic activity, inhibits platelet aggregation, has antioxidant properties, improves vascular endothelial function, inhibits the increase of monocyte chemotactic factor protein levels and the aggregation of monocytes and macrophages in ischemic areas, and has effects on vascular smooth muscle cells.
[0003] Research indicates that the potency of urokinase is primarily determined using the substrate method. The potency of urokinase is defined as "the amount of urokinase that hydrolyzes 1 μmol of HD-valine-L-leucine-L-arginine-P-p-nitroaniline hydrochloride (HD-Val-Leu-Arg-PNA·2HCl) in 1 minute at 37°C and pH 8.0 is 1 PNA unit." This is because urokinase can hydrolyze the specific chromogenic substrate HD-Val-Leu-Arg-PNA·2HCl to generate the hydrolysis product p-nitroaniline (PNA). The specific experimental steps are as follows:
[0004] (1) Solution preparation
[0005] To prepare a 0.2 mol / L tris(hydroxymethyl)aminomethane buffer solution, dissolve 24.2 g of tris(hydroxymethyl)aminomethane in 800 ml of water, adjust the pH to 8.0 with 6 mol / L hydrochloric acid solution, and dilute with water to 1000 ml.
[0006] For the substrate solution, take one vial of the chromogenic substrate S-2266 (equivalent to 25 mg of HD-Val-Leu-Arg-PNA·2HCl), dissolve and dilute it with water to prepare a 1.5 mmol / L solution. Store at low temperature.
[0007] Take an appropriate amount of this product and dilute it quantitatively with 0.2 mol / L tris(hydroxymethyl)aminomethane buffer to prepare a solution containing approximately 0.03 PNA units per ml.
[0008] (2) Measurement method
[0009] Take four test tubes and add 4.0 ml of 0.2 mol / L tris(hydroxymethyl)aminomethane buffer to each. Incubate in a water bath at 37℃±0.5℃ for 5 minutes. Then, accurately add 0.2 ml of the test solution to each tube, mix well, and incubate in a water bath at 37℃±0.5℃ for 5 minutes. Next, add 0.4 ml of 50% acetic acid solution to the first tube (as a blank reaction tube) and 0.4 ml of substrate solution to the remaining three tubes (as test sample tubes). Shake well immediately and start timing. Incubate in a water bath at 37℃±0.5℃ for a total of 15 minutes. Then, add 0.4 ml of substrate solution to the first tube and 0.4 ml of 50% acetic acid solution to each of the second, third, and fourth tubes. Measure the absorbance at a wavelength of 405 nm. The absorbance should be controlled between 0.1 and 0.2. Calculate using the following formula:
[0010] The potency of 1 ml of urokinase (PNA units) = 173.6 × ((At1 + At2 + At3) / 3 - At0) × T / 1000
[0011] In the formula, 173.6 is the reaction constant; At0 is the absorbance of the blank reaction tube; At1-At3 are the absorbances of the test sample tube; T is the dilution factor; and 1000 is the conversion value between L and ml.
[0012] However, the method has the following shortcomings:
[0013] 1. Large analytical error: According to the requirements of the General Chapter 0401 of the Pharmacopoeia, Volume IV, on ultraviolet-visible spectrophotometry, "the absorbance reading of the test solution should generally be between 0.3 and 0.7 Abs." When the absorbance is below 0.3 Abs, the detection results are greatly affected by instrument noise, resulting in a large analytical error. In the original method, the sample absorbance was approximately 0.18 Abs, and a fluctuation of 0.002 Abs in absorbance would lead to a 1% relative error.
[0014] 2. The original method lacks a standard for comparison, and the test results are highly dependent on the reaction conditions. Previous studies have shown that the pH value of the buffer solution should be controlled between 7.95 and 8.10, the test process should be free from airflow (such as air conditioning vents), and a covered water bath should be used. Environmental factors can cause significant errors.
[0015] 3. Low detection efficiency: The original method uses ultraviolet spectrophotometer, and sample readings are taken sequentially using cuvettes. The operation is cumbersome and time-consuming, with sample analysis time of about 1.5 hours per sample.
[0016] Therefore, developing a simple and more stable method for valence detection can help improve detection efficiency and result reliability, which is of great significance to product quality research. Summary of the Invention
[0017] This invention provides an accurate, reliable, and efficient method for detecting the potency of urokinase.
[0018] This invention provides a method for detecting the potency of urokinase, comprising the following steps:
[0019] (i) Solution preparation:
[0020] Substrate solution: Prepare a substrate solution of 0.12-0.30 mmol / L by adding water to the chromogenic substrate HD-Val-Leu-Arg-PNA·2HCl;
[0021] Standard solutions: Prepare gradient concentration solutions of urokinase standard in 0.15-0.25 mol / L Tris buffer solution with pH 7.5-8.5 to construct a standard curve;
[0022] Test solution: Prepare a test solution by dissolving the test sample in 0.15-0.25 mol / L Tris buffer solution with a pH of 7.5-8.5;
[0023] (ii) Take a 96-well plate, accurately transfer the standard solution of each concentration and the test solution into each well of the 96-well plate, then transfer the substrate solution into each well, mix well, react at 37±1℃ for 14-18 min, and read the absorbance of each well at a wavelength of 405±10nm on an ELISA reader.
[0024] (iii) Plot a standard curve with the concentration of the standard as the x-axis and the absorbance of the standard as the y-axis. Substitute the absorbance of the test sample into the standard curve to calculate the concentration of the test sample.
[0025] In another preferred embodiment, the concentration of the chromogenic substrate HD-Val-Leu-Arg-PNA·2HCl in the substrate solution is 0.14-0.5 mmol / L, preferably 0.15-0.2 mmol / L.
[0026] In another preferred embodiment, the Tris buffer solution used for the standard solution and the test solution may have the same or different pH and concentration, preferably the same.
[0027] In another preferred embodiment, the pH of the Tris buffer solution is 7.8-8.2, preferably 8.0-8.1.
[0028] In another preferred embodiment, the concentration of the Tris buffer solution is 0.18-0.22 mol / L, preferably 0.2-0.21 mol / L.
[0029] In another preferred embodiment, the standard solution with gradient concentrations has at least 5 gradients, preferably 5-7 gradients.
[0030] In another preferred embodiment, the gradient concentration range of the standard solution is preferably 0.008-0.05 PNA units / ml, more preferably 0.01-0.03 PNA units / ml.
[0031] In another preferred embodiment, the concentration of the test solution is within the gradient concentration range of the standard solution, preferably 0.01-0.03 PNA units / ml.
[0032] In another preferred embodiment, the R of the standard curve 2 ≥0.99.
[0033] In another preferred embodiment, in step (ii), the volume ratio of the standard solution or test solution added to each well to the volume of the substrate solution is 1:0.8-1.2, preferably 1:0.9-1.1, and more preferably 1:1.
[0034] In another preferred embodiment, in step (ii), the volume of the standard solution or test solution added to each well is 0.08-0.2 ml, preferably 0.1-0.12 ml.
[0035] In another preferred embodiment, in step (ii), the volume of substrate solution added to each well is 0.08-0.2 ml, preferably 0.1-0.12 ml.
[0036] In this invention, the well treatment methods for standard products and test samples from the same batch are kept consistent.
[0037] In another preferred embodiment, no incubation time is required after sample addition according to the present invention.
[0038] In another preferred embodiment, no termination step is required after the reaction time of the present invention has elapsed (e.g., no need to add acetic acid to terminate the reaction).
[0039] In another preferred embodiment, the detection time of the present invention is ≤1h.
[0040] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description
[0041] Figure 1 The linear relationship between concentration and absorbance was shown at a substrate concentration of 0.075 mmol / L.
[0042] Figure 2 The linear relationship between concentration and absorbance was shown at a substrate concentration of 0.15 mmol / L.
[0043] Figure 3The linear relationship between concentration and absorbance was shown at a substrate concentration of 0.3 mmol / L.
[0044] Figure 4 The linear relationship between concentration and absorbance was shown when the reaction time was 10 min.
[0045] Figure 5 The linear relationship between concentration and absorbance was shown when the reaction time was 15 min.
[0046] Figure 6 The linear relationship between concentration and absorbance was shown when the reaction time was 20 min.
[0047] Figure 7 The linear relationship between the concentration and absorbance of the buffer solution at pH 6.0 was shown.
[0048] Figure 8 The linear relationship between the concentration and absorbance of the buffer solution at pH 8.0 was shown.
[0049] Figure 9 The linear relationship between the concentration and absorbance of the buffer solution at pH 9.0 was shown.
[0050] Figure 10 This is the standard curve for Example 2. Detailed Implementation
[0051] Through extensive and in-depth research, and through numerous screenings and tests, the inventors have developed a method for detecting urokinase potency that is simpler to operate, faster to detect, and provides more accurate and stable results. By optimizing the substrate concentration and using a microplate reader for reading, the absorbance of the test sample is reduced to approximately 0.4 Abs, thus minimizing instrument detection error. Furthermore, by introducing standards and reducing the number of experimental steps, operational errors are reduced. Using a 96-well plate for reaction and reading significantly improves detection efficiency, with sample analysis time of approximately 1 hour per plate. This invention is based on these findings.
[0052] the term
[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0054] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
[0055] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.
[0056] As used herein, the terms “room temperature” or “normal temperature” refer to a temperature of 4-40°C, preferably 25±5°C.
[0057] Methods for detecting the potency of urokinase
[0058] This invention provides a method for detecting the potency of urokinase, comprising the following steps:
[0059] (i) Solution preparation:
[0060] Substrate solution: Prepare a substrate solution of 0.12-0.30 mmol / L by adding water to the chromogenic substrate HD-Val-Leu-Arg-PNA·2HCl;
[0061] Standard solutions: Prepare gradient concentration solutions of urokinase standard in 0.15-0.25 mol / L Tris buffer solution with pH 7.5-8.5 to construct a standard curve;
[0062] Test solution: Prepare a test solution by dissolving the test sample in 0.15-0.25 mol / L Tris buffer solution with a pH of 7.5-8.5;
[0063] (ii) Take a 96-well plate, accurately transfer the standard solution of each concentration and the test solution into each well of the 96-well plate, then transfer the substrate solution into each well, mix well, react at 37±1℃ for 14-18 min, and read the absorbance of each well at a wavelength of 405±10nm on an ELISA reader.
[0064] (iii) Plot a standard curve with the concentration of the standard as the x-axis and the absorbance of the standard as the y-axis. Substitute the absorbance of the test sample into the standard curve to calculate the concentration of the test sample.
[0065] In another preferred embodiment, the concentration of the chromogenic substrate HD-Val-Leu-Arg-PNA·2HCl in the substrate solution is 0.14-0.5 mmol / L, preferably 0.15-0.2 mmol / L.
[0066] In another preferred embodiment, the Tris buffer solution used for the standard solution and the test solution may have the same or different pH and concentration, preferably the same.
[0067] In another preferred embodiment, the pH of the Tris buffer solution is 7.8-8.2, preferably 8.0-8.1 (which can be adjusted with hydrochloric acid).
[0068] In another preferred embodiment, the concentration of the Tris buffer solution is 0.18-0.22 mol / L, preferably 0.2-0.21 mol / L.
[0069] In another preferred embodiment, the standard solution with gradient concentrations has at least 5 gradients, preferably 5-7 gradients.
[0070] In another preferred embodiment, the gradient concentration range of the standard solution is preferably 0.008-0.05 PNA units / ml, more preferably 0.01-0.03 PNA units / ml.
[0071] In another preferred embodiment, the concentration of the test solution is within the gradient concentration range of the standard solution, preferably 0.01-0.03 PNA units / ml.
[0072] In another preferred embodiment, the R of the standard curve 2 ≥0.99.
[0073] In another preferred embodiment, in step (ii), the volume ratio of the standard solution or test solution added to each well to the volume of the substrate solution is 1:0.8-1.2, preferably 1:0.9-1.1, and more preferably 1:1.
[0074] In another preferred embodiment, in step (ii), the volume of the standard solution or test solution added to each well is 0.08-0.2 ml, preferably 0.1-0.12 ml.
[0075] In another preferred embodiment, in step (ii), the volume of substrate solution added to each well is 0.08-0.2 ml, preferably 0.1-0.12 ml.
[0076] In this invention, the well treatment methods for standard products and test samples from the same batch are kept consistent.
[0077] In another preferred embodiment, no incubation time is required after sample addition according to the present invention.
[0078] In another preferred embodiment, no termination step is required after the reaction time of the present invention is completed (e.g., no acetic acid is needed to terminate the reaction, whereas in the original method system, due to the slow detection speed, the detection time of each tube is different, and acetic acid needs to be added to terminate the reaction to avoid inconsistent reaction time).
[0079] In another preferred embodiment, the single-batch detection time of the present invention is ≤1 hour. Multiple samples can be detected simultaneously in one batch, depending on the capacity of the 96-well plate, such as 1-45 samples, preferably 5-45 samples.
[0080] The main advantages of this invention include:
[0081] 1. By using 96-well plates instead of test tubes, the overall reaction system is optimized from 5 ml in the original method to 0.2 ml. The reaction process is optimized to mix the sample and substrate solution and react at 37°C for 15 min, with the readings taken by a microplate reader. This reduces the steps of 5 min buffer incubation and acetic acid termination in the original method, thus reducing the detection time (the detection time of this invention is approximately 1 h / plate (including sample pretreatment time; the microplate reader can take unified readings of samples on the 96-well plate at the same time, allowing for the simultaneous detection of approximately 45 samples)). Compared to the original method's 1.5 h / sample, this significantly improves detection efficiency and is more economical.
[0082] 3. The introduction of Urokinase standard to create a standard curve for detection improves the accuracy and reliability of the detection and reduces the instability of experimental results caused by reaction conditions and the operator's operation.
[0083] 4. At the same sample concentration, the absorbance reading is significantly improved (e.g., the absorbance reading of a sample with 0.02 PNA units / ml is approximately 0.4 Abs, and a fluctuation of 0.004 Abs in absorbance leads to a 1% relative error, while the original method's absorbance reading of a sample with 0.03 PNA units / ml is approximately 0.18 Abs, and a fluctuation of 0.002 Abs in absorbance leads to a 1% relative error). This reduces instrument reading errors. Furthermore, method validation data show that this method has good linearity, repeatability, intermediate precision, and accuracy, and the repeatability, intermediate precision, and accuracy results are better than the original method. This indicates that the results of this method are reliable and accurate.
[0084] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.
[0085] Example 1
[0086] The experimental procedure is as follows:
[0087] 3.1 Solution preparation:
[0088] 6 mol / L hydrochloric acid solution: Take 50 ml of concentrated hydrochloric acid, dilute with purified water to 100 ml, mix well, and it is ready.
[0089] 0.2 mol / L Tris (Tris) buffer: Dissolve 24.2 g of Tris in 800 ml of purified water, adjust the pH to about 8.0 with 6 mol / L hydrochloric acid solution, add purified water to a final volume of 1000 ml, and shake well.
[0090] Substrate solution: Take one vial of chromogenic substrate S-2266 (equivalent to 25 mg of HD-Val-Leu-Arg-PNA·2HCl), dissolve it in 14.4 ml of water to prepare a stock solution containing 3 mmol / L of S-2266. After aliquoting, store at -20°C or below. Before use, dilute 20 times with purified water to prepare an application solution of 0.15 mmol / L.
[0091] Standard solutions: Take one vial of Urokinase standard and dilute it with 0.2 mol / L Tris buffer to prepare solutions of 0.010, 0.015, 0.020, 0.025, and 0.030 PNA units / ml, which will be used as the standard curve.
[0092] Test solution: Take the test sample and dilute it with 0.2 mol / L Tris buffer to prepare a solution with a concentration of 0.020 PNA units / ml, which is used as the test solution.
[0093] 3.2 Testing Procedures
[0094] Take a 96-well plate and accurately transfer 0.1 ml of each concentration of standard solution and test solution into each well of the 96-well plate. Then transfer 0.1 ml of substrate solution into each of the above wells. Mix well immediately and react at 37°C for 15 min. Read the absorbance of each well at a wavelength of 405 nm using a microplate reader (Multiskan ET, Thermo Fisher Scientific).
[0095] 3.3 Calculation
[0096] Plot a standard curve (R0) with the concentration of the standard on the x-axis and the absorbance of the standard on the y-axis. 2 (≥0.99), the absorbance of the test sample is substituted into the standard curve to calculate the concentration of the test sample.
[0097] 3.4 Method Exploration
[0098] 3.4.1 Preparation of Standards
[0099] Since there were no commercially available standards for potency testing of urokinase, we used urokinase active pharmaceutical ingredient (batch number: 241207S01) produced by our company to prepare the experimental standard. All test items of this batch of active pharmaceutical ingredient met the national drug standards for urokinase after full inspection. Furthermore, the purity of this batch of active pharmaceutical ingredient was 100%, and its amino acid sequence was consistent with the theoretical sequence of urokinase.
[0100] According to the formulation in Table 1, a standard for urokinase (batch number: 241227P01) was prepared from the active pharmaceutical ingredient of this batch, and a full inspection was performed on it (results are shown in Table 2). All test indicators of the prepared standard met the national drug standards for urokinase for injection. The potency calibration results of two laboratory technicians are shown in Table 3 below. The average value of twelve results was 0.150 PNA units / vial, and the RSD value was 1.7%. Therefore, the labeled amount of the standard was set at 0.15 PNA units / vial. The stability of the standard was continuously investigated by placing it at 5℃±3℃.
[0101] Table 1. Prescription
[0102]
[0103]
[0104] Table 2. Quality test results of Uriagene standard products
[0105]
[0106] Table 3. Results of Potency Calibration of Urokinase Standard (Original Potency Testing Method)
[0107]
[0108] 3.4.2 Selection of substrate concentration
[0109] (1) Preparation of substrate solution: Take one vial of chromogenic substrate S-2266 (equivalent to 25 mg of HD-Val-Leu-Arg-PNA·2HCl), add 14.4 ml of water to dissolve it, and prepare a stock solution with a concentration of 3 mmol / L of S-2266. After aliquoting, store at -20℃ or below. Before use, dilute with purified water to prepare application solutions of 0.075, 0.15, and 0.3 mmol / L respectively.
[0110] (2) Experimental steps
[0111] Perform the detection using substrate solutions of different concentrations according to the "3.2 Detection Procedure".
[0112] (3) Experimental Results
[0113] The test results are shown in Table 5 below. The results indicate that substrate concentrations of 0.15 mmol / L and 0.3 mmol / L react sufficiently with the standards at various concentrations, yielding good linearity. Considering both the testing cost and the test results, a substrate concentration of 0.15 mmol / L is the most suitable.
[0114] Table 5. Results of standard curves using different substrate concentrations
[0115]
[0116] 3.4.3 Selection of reaction time
[0117] (1) Experimental steps
[0118] Take a 96-well plate and accurately transfer 0.1 ml of each concentration of standard solution and test solution into each well of the 96-well plate. Then transfer 0.1 ml of substrate solution into each of the above wells. Mix well immediately and react in an ELISA reader at 37°C for 10, 15 and 20 minutes respectively. Read the absorbance at a wavelength of 405 nm.
[0119] (2) Test results
[0120] The test results are shown in Table 6 below. The linearity results show that when the reaction time is 15 min, the standard curve R... 2 Since the value is ≥0.99, the final reaction time chosen was 15 min.
[0121] Table 6. Results of standard curves for different reaction times
[0122]
[0123]
[0124] 3.4.3 Investigation of the effect of buffer pH
[0125] (1) Take 0.2 mol / L Tris buffer solution and adjust it to pH 6.0, 8.0 and 9.0 respectively. Dilute the standard with buffer solution of different pH and perform detection according to the detection steps in 3.2 to investigate the effect of buffer solution of different pH on the detection results.
[0126] (2) Experimental Results
[0127] The test results are shown in Table 7 below; according to the test results, the pH of the buffer solution should be 8.0.
[0128] Table 7. Effect of buffer pH on detection results
[0129]
[0130]
[0131] 3.4.4 Summary
[0132] After examining the experimental conditions, the final experimental settings were determined to be a substrate concentration of 0.15 mmol / L, a reaction time of 15 min, and a buffer pH of 8.0.
[0133] Example 2
[0134] 3.5 Methodological Validation
[0135] 3.5.1 Linear
[0136] (1) Take one vial of Urokinase standard (241227P01) and dilute it with 0.2 mol / L Tris buffer to prepare solutions of 0.010, 0.015, 0.020, 0.025, and 0.030 PNA units / ml as the standard curve. Perform the detection according to the detection procedure in 3.2.
[0137] (2) Experimental Results
[0138] From Table 8 and Figure 10 It can be seen that the R of the standard curve 2 The linearity was ≥0.99, indicating good linearity; and the RSD values of the standard curve for each concentration point, repeated three times, were all less than 3.0%, indicating good repeatability; the linear recoveries were all between 95.0% and 105.0%. In summary, this method exhibits good linearity.
[0139] Table 8. Standard Curve Data
[0140]
[0141]
[0142] 3.5.2 Repeatability
[0143] (1) Analyst 1 took the test sample and diluted it with 0.2 mol / L Tris buffer to prepare a solution of 0.020 PNA units / ml. The sample was measured 6 times according to the detection method in 3.2.
[0144] (2) Experimental Results
[0145] Table 9 shows the repeatability test results of the test sample. It can be seen that the RSD value of the test results is 1.35%. Compared with the repeatability results of the original potency method, the results of the two test methods are similar, but the RSD value of the repeatability of this method is smaller, so the repeatability of this method is better.
[0146] Table 9. Repeatability test results
[0147]
[0148]
[0149] 3.5.3 Intermediate Precision
[0150] (1) Analyst 2 took the test sample and diluted it with 0.2 mol / L Tris buffer to prepare a solution of 0.020 PNA units / ml. The sample was measured 6 times according to the detection method in 3.2.
[0151] (2) Experimental Results
[0152] The test results are shown in Table 10. The RSD value of the test results of the same batch of samples was 1.23%. The RSD of the 12 results from the two inspectors was 1.27%. Compared with the intermediate precision results of the original potency method, the RSD value of the intermediate precision of this method was smaller and the intermediate precision was better.
[0153] Table 10. Intermediate Precision Results
[0154]
[0155]
[0156] 3.5.4 Accuracy
[0157] (1) Use the 221212S01 batch sample to perform spiking recovery at three concentrations of 80%, 100%, and 120%, and perform the detection according to the detection method in 3.2;
[0158] (2) Experimental Results
[0159] The recovery rates for low, medium, and high concentrations are shown in Table 11 below. The recovery rates are all between 95% and 105%. Compared with the accuracy results of the original potency method, the recovery rate results of this method are better, proving that the method is more accurate.
[0160] Table 11. Accuracy Results
[0161]
[0162]
[0163] 3.6 Summary
[0164] Based on the above methodological verification results, it can be seen that the potency detection method of the present invention has good linearity, repeatability, intermediate precision and accuracy, and the repeatability, intermediate precision and accuracy are better than the original detection method, and the results are more stable and reliable.
[0165] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.
Claims
1. A method for detecting the potency of urokinase, characterized in that, Including the following steps: (i) Solution preparation: Substrate solution: Prepare a substrate solution of 0.12-0.30 mmol / L by adding water to the chromogenic substrate HD-Val-Leu-Arg-PNA·2HCl; Standard solutions: Prepare gradient concentration solutions of urokinase standard in 0.15-0.25 mol / L Tris buffer solution with pH 7.5-8.5 to construct a standard curve; Test solution: Prepare a test solution by dissolving the test sample in 0.15-0.25 mol / L Tris buffer solution with a pH of 7.5-8.5; (ii) Take a 96-well plate, accurately transfer the standard solution of each concentration and the test solution into each well of the 96-well plate, then transfer the substrate solution into each well, mix well, react at 37±1℃ for 14-18 min, and read the absorbance of each well at a wavelength of 405±10nm on an ELISA reader. (iii) Plot a standard curve with the concentration of the standard as the x-axis and the absorbance of the standard as the y-axis. Substitute the absorbance of the test sample into the standard curve to calculate the concentration of the test sample.
2. The method as described in claim 1, characterized in that, The concentration of the chromogenic substrate HD-Val-Leu-Arg-PNA·2HCl in the substrate solution is 0.14-0.5 mmol / L, preferably 0.15-0.2 mmol / L.
3. The method as described in claim 1, characterized in that, The pH of the Tris buffer solution is 7.8-8.2, preferably 8.0-8.
1.
4. The method as described in claim 1, characterized in that, The concentration of the Tris buffer solution is 0.18-0.22 mol / L, preferably 0.2-0.21 mol / L.
5. The method as described in claim 1, characterized in that, The standard solution with gradient concentrations has at least 5 gradients, preferably 5-7 gradients.
6. The method as described in claim 1, characterized in that, The gradient concentration range of the standard solution is preferably 0.008-0.05 PNA units / ml, and more preferably 0.01-0.03 PNA units / ml.
7. The method as described in claim 1, characterized in that, The standard curve R 2 ≥0.
99.
8. The method as described in claim 1, characterized in that, In step (ii), the volume ratio of the standard solution or test solution added to each well to the volume of the substrate solution is 1:0.8-1.2, preferably 1:0.9-1.1, and more preferably 1:
1.
9. The method as described in claim 1, characterized in that, In step (ii), the volume of the standard solution or test solution added to each well is 0.08-0.2 ml, preferably 0.1-0.12 ml.
10. The method as described in claim 1, characterized in that, In step (ii), the volume of substrate solution added to each well is 0.08-0.2 ml, preferably 0.1-0.12 ml.