Method for determining titer of lumbrukinase

Abstract

The present invention relates to the technical field of lumbrukinase titer determination, particularly to a lumbrukinase titer determination method, which comprises: (1) taking a lumbrukinase test sample and a lumbrukinase standard substance, and respectively dissolving with a sodium chloride solution to obtain a test sample solution and standard substance solutions with different concentrations; (2) taking a fibrinogen solution, adding an agarose solution and a thrombin solution, uniformly mixing, quickly pouring into an elisa plate, and determining the OD value of each hole by using a microplate reader after agar is solidified; respectively adding the test solution and the standard solution into different holes, enabling the lumbrukinase to react with the fibrin, and determining the OD value of each hole by using a microplate reader after the reaction is finished; and calculating the OD value change difference rate. And calculating a regression equation according to the titer of the lumbrukinase standard substance and the OD value change difference rate, substituting the OD measured value of the test sample into the regression equation, and calculating the titer of the test sample. The method has the advantages of short time consumption, high correlation, strong specificity, high detection precision and high accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of lumbrokinase titer determination, and in particular to a method for determining lumbrokinase titer. Background Art

[0002] Lumbrukinase is a group of proteolytic enzymes extracted from special earthworms, with a molecular weight of 16,000-45,000 Da. Lumbrukinase is an antithrombotic drug, which is mainly used clinically for thrombotic diseases, especially for patients with increased fibrinogen and platelet aggregation rate; it can be used for ischemic cardiovascular and cerebrovascular diseases to improve symptoms and prevent the progression of the disease.

[0003] Currently, the potency of lumbrokinase is determined using the standard of "WS1-(X-052)-2001Z-2010". This method uses manual drilling and manual measurement, which is subject to greater interference from human factors and has the following defects: the size of the dissolution zone is easily affected by the depth of the drilling, and the depth of the hole is inversely proportional to the diameter of the dissolution zone; due to the boundary effect of the diameter of the dissolution zone, there is a 5%-10% error in the measurement; since drilling and measurement of the dissolution zone are both manual operations, there are large and unavoidable measurement errors; the reaction time is long, and the potency can only be determined after 18 hours of reaction.

[0004] Chinese patent application CN201711166331.2 discloses a method for determining the potency of lumbrokinase in earthworm extract intermediates by near-infrared spectroscopy. However, this method has low correlation and weak specificity, and also requires 18 hours of reaction before the potency can be determined.

[0005] Therefore, it is very necessary to develop a method for determining the potency of lumbrokinase with high automation, good accuracy and fast detection speed. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a new instrumental analysis method for determining the potency of lumbrokinase with short time, strong specificity, high detection precision and accuracy.

[0007] The present invention uses an ELISA instrument to measure the absorbance value of agar gel in a 96-well ELISA plate before and after spotting. The difference in the absorbance value of agar gel before and after spotting on the 96-well ELISA plate is used to reflect the degree of fibrinolysis by lumbrokinase. The greater the difference in absorbance value before and after, the deeper the degree of fibrinolysis by lumbrokinase, indicating that the lumbrokinase titer is higher. This principle is used to quantitatively determine the lumbrokinase titer.

[0008] The present invention is achieved through the following technical solutions: A method for quantitatively determining the potency of lumbrokinase based on the change in the absorbance value of agar gel before and after the reaction of fibrin and lumbrokinase, comprising the following steps: (1) Take the lumbrokinase test sample and the lumbrokinase standard, dissolve them in sodium chloride solution respectively to obtain the test sample solution and the standard solution of different concentrations; (2) Take the fibrinogen solution, add the agarose solution and the thrombin solution, mix well and quickly pour into a 96-well ELISA plate, and measure the OD value of each well with an ELISA reader, which is recorded as the OD blank; add the test solution and the standard solution to different wells to allow lumbrokinase to react with fibrinogen. After the reaction is completed, measure the OD value of each well with an ELISA reader, which is recorded as the OD measurement; calculate the OD value change difference rate according to the following formula: ; The difference rate of change in OD value is correlated with the potency of the lumbrokinase standard. The regression equation is calculated based on the potency of the lumbrokinase standard and the difference rate of change in OD value, and the OD measurement value of the test sample is substituted into the regression equation to calculate the potency of the test sample.

[0009] In some embodiments of the present invention, the reaction time in step (2) is 5-6 hours.

[0010] In some embodiments of the present invention, the detection wavelength of the microplate reader in step (2) is 490 nm.

[0011] In some embodiments of the present invention, in step (2), the concentration of the fibrinogen solution is 1.4-1.6 mg / mL, the concentration of the agarose solution is 1.4-1.6 g / 100 mL, and the concentration of the thrombin solution is 0.9-1.1 BP / mL.

[0012] In some embodiments of the present invention, the volume ratio of the fibrinogen solution, the agarose solution and the thrombin solution in step (2) is 13:13:1.

[0013] In some embodiments of the present invention, the mass concentration of the sodium chloride solution in step (1) is 0.9%.

[0014] In some embodiments of the present invention, the concentration of the standard solution in step (1) is 10000 IU, 9000 IU, 8000 IU, 7000 IU, 6000 IU, 5000 IU, and 4000 IU lumbrokinase units per 1 mL of the standard solution, respectively.

[0015] In some embodiments of the present invention, the temperature of the agarose solution in step (2) is 50-60°C.

[0016] In some embodiments of the present invention, before measuring the OD value of each well with an ELISA reader in step (2), the wells are placed at room temperature (20-30° C.) for 0.5-1 h.

[0017] In some embodiments of the present invention, the reaction temperature in step (2) is 36-38°C.

[0018] The present invention changes the method of determining the potency of lumbrokinase by manually measuring the size of the lysis zone, and introduces a new method of instrumental analysis. The measurement is performed using an ELISA instrument, and the measurement data is more stable and accurate. The potency is quantitatively determined by measuring the difference in the absorbance value of the agar gel in the 96-well ELISA plate before and after spotting by a method in which visible light penetrates the ELISA wells in three dimensions. The present invention quantitatively determines the potency of lumbrokinase based on the change in the absorbance value of the agar gel before and after the reaction of fibrin and lumbrokinase, and is different from the simple ultraviolet-visible spectrophotometry method for determining the absorbance value. During the ultraviolet-visible spectrophotometry determination process, other proteins also absorb at the specific absorption wavelength of lumbrokinase, resulting in inaccurate measurement data and poor specificity. The present invention effectively avoids interference from human factors and technical defects, and also avoids the problem of weak specificity of ultraviolet spectrophotometry.

[0019] The beneficial effects of the present invention are: 1. The present invention solves the error problems of manual punching and manual measurement in the original standard "WS1-(X-052)-2001Z-2010", and effectively improves the inspection precision.

[0020] 2. The present invention avoids the method of calculating the potency of lumbrokinase by manually measuring the size of the lysis zone. All data are objectively given by the instrument, avoiding the errors caused by human factors in the detection process, improving the objectivity of the method, and effectively improving the accuracy of the test.

[0021] 3. The present invention solves the problem of long detection time in the original standard method, effectively shortens the detection time, and can complete the reaction within 6 hours, thereby improving the detection efficiency.

[0022] 4. The potency results measured by the present invention are similar to those of the original standard method, but the required detection time is shortened from 18 hours to less than 6 hours, which is shortened by more than two thirds; the RSD value of the detection precision is reduced from 5.4% to 1.9%, and the detection precision is improved by more than 60%. The linear R 2 The value is greater than 0.990, indicating a good linear relationship.

[0023] 5. The present invention quantitatively determines the potency of lumbrokinase based on the change in the absorbance value of agar gel before and after the reaction of fibrin and lumbrokinase. There is no interference from other proteins, the measurement data is more accurate, and the specificity is strong. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the standard curve of Example 1 of the present invention.

[0025] Figure 2 It is the standard curve of Example 2 of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0027] Example 1 1.1 Reagents and standards: Reagents: disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, agarose, fibrinogen, thrombin; Standard: Lumbrokinase standard.

[0028] 1.2 Operation: 1.2.1 Preparation of reagents: 0.01 mol / L phosphate buffer (pH 7.8): Take 3.58 g of disodium hydrogen phosphate, add water to dissolve and dilute to 1000 mL to form solution A; take 0.78 g of sodium dihydrogen phosphate (NaH2PO4·2H2O), add water to dissolve and dilute to 500 mL to form solution B; mix solutions A and B until the pH value reaches 7.8.

[0029] 0.9% sodium chloride solution: Take 9 g of sodium chloride (NaCl), add water to dissolve and dilute to 1000 mL.

[0030] Working solution: Mix 0.01 mol / L phosphate buffer (pH 7.8) and 0.9% sodium chloride solution (1:17, V / V).

[0031] Agarose solution: Take 1.4 g of agarose and add 100 mL of working solution and heat to dissolve.

[0032] Fibrinogen solution: Take an appropriate amount of fibrinogen and add working solution to make a solution containing 1.4 mg of coagulable protein per 1 mL. Fibrinogen was purchased from China Food and Drug Inspection Institute, catalog number 140607-202244.

[0033] Thrombin solution: Take thrombin and add 0.9% sodium chloride solution to make a solution containing 0.9 BP units per 1 mL. Thrombin was purchased from China Food and Drug Inspection Institute, catalog number 140605-202228.

[0034] 1.2.2 Preparation of standard solution: Lumbrokinase standard was taken and prepared with 0.9% sodium chloride solution to prepare solutions with concentrations of 10000 IU, 9000 IU, 8000 IU, 7000 IU, 6000 IU, 5000 IU, and 4000 IU lumbrokinase units per 1 mL. Lumbrokinase standard was purchased from China Food and Drug Inspection Institute with the catalog number 140650-201804.

[0035] 1.2.3 Preparation of test solution: Take an appropriate amount of lumbrokinase test sample, add 0.9% sodium chloride solution to dissolve it and dilute it to a concentration within the range of the standard curve.

[0036] 1.3 Determination: Take 130 mL of fibrinogen solution, place it in a beaker, add 130 mL of 50℃ agarose solution and 10 mL of thrombin solution while stirring, mix immediately, quickly pour into a 96-well ELISA plate, and place horizontally at room temperature for 0.5 hours to allow the mixed solution to solidify. Measure the OD value of each well with an ELISA reader at a wavelength of 490 nm, exclude the wells with large OD value deviation, accurately measure 20 µL of lumbrokinase standard solution and test solution of different concentrations, and add them to different assay wells of the same ELISA plate, so that the titer of the lumbrokinase standard in the assay well is 200 IU, 180 IU, 160 IU, 140 IU, 120 IU, 100 IU, and 80 IU, and measure the OD value of each well with an ELISA reader at a wavelength of 490 nm (recorded as OD blank). Cover and place in a 36℃ incubator for reaction for 5 hours. After taking out, the OD value was measured at 490 nm using an enzyme marker (referred to as OD measurement), and the OD value change difference rate was calculated according to the following formula.

[0037] .

[0038] The standard curve was obtained by taking the potency of the lumbrokinase standard as the horizontal axis and the OD value change difference rate as the vertical axis. Figure 1 As shown, the regression equation is calculated and the regression equation is obtained: Y = 0.001547*X + 0.4213, R 2 =0.9979.

[0039] Substitute the difference rate of the OD value of the test sample into the regression equation to calculate the number of test sample potency units. Six wells should be made for each standard sample and test sample, and the average value should be used for calculation.

[0040] Example 2 1.1 Reagents and standards: Reagents: disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, agarose, fibrinogen, thrombin; Standard: Lumbrokinase standard.

[0041] 1.2 Operation: 1.2.1 Preparation of reagents: 0.01 mol / L phosphate buffer (pH 7.8): Take 3.58 g of disodium hydrogen phosphate, add water to dissolve and dilute to 1000 mL to form solution A; take 0.78 g of sodium dihydrogen phosphate (NaH2PO4·2H2O), add water to dissolve and dilute to 500 mL to form solution B; mix solutions A and B until the pH value reaches 7.8.

[0042] 0.9% sodium chloride solution: Take 9 g of sodium chloride (NaCl), add water to dissolve and dilute to 1000 mL.

[0043] Working solution: Mix 0.01 mol / L phosphate buffer (pH 7.8) and 0.9% sodium chloride solution (1:17, V / V).

[0044] Agarose solution: Take 1.6 g of agarose and add 100 mL of working solution and heat to dissolve.

[0045] Fibrinogen solution: Take an appropriate amount of fibrinogen and add working solution to make a solution containing 1.6 mg of coagulable protein per 1 mL. Fibrinogen was purchased from China Food and Drug Inspection Institute, catalog number 140607-202244.

[0046] Thrombin solution: Take thrombin and add 0.9% sodium chloride solution to make a solution containing 1.1 BP units per 1 mL. Thrombin was purchased from China Food and Drug Inspection Institute, catalog number 140605-202228.

[0047] 1.2.2 Preparation of standard solution: Lumbrokinase standard was taken and prepared with 0.9% sodium chloride solution to prepare solutions with concentrations of 10000 IU, 9000 IU, 8000 IU, 7000 IU, 6000 IU, 5000 IU, and 4000 IU lumbrokinase units per 1 mL. Lumbrokinase standard was purchased from China Food and Drug Inspection Institute with the catalog number 140650-201804.

[0048] 1.2.3 Preparation of test solution: Take an appropriate amount of lumbrokinase test sample, add 0.9% sodium chloride solution to dissolve it and dilute it to a concentration within the range of the standard curve.

[0049] 1.3 Determination: Take 130 mL of fibrinogen solution, place it in a beaker, add 130 mL of 60℃ agarose solution and 10 mL of thrombin solution while stirring, mix immediately, quickly pour into a 96-well ELISA plate, and place horizontally at room temperature for 1 hour to allow the mixed solution to solidify. Use an ELISA reader to measure the OD value of each well at a wavelength of 490nm, remove the wells with large OD value deviations, accurately measure 20 µL of lumbrokinase standard solution and test solution of different concentrations, and add them to different assay wells of the same ELISA plate, so that the titer of the lumbrokinase standard in the assay well is 200 IU, 180 IU, 160 IU, 140 IU, 120 IU, 100 IU, and 80 IU, and use an ELISA reader to measure the OD value of each well at a wavelength of 490 nm (recorded as OD blank). Cover and place in a 38℃ incubator for reaction for 6 hours. After taking out, use an ELISA reader to measure the OD value at 490 nm (recorded as OD determination), and calculate the difference rate of OD value change according to the following formula.

[0050] .

[0051] The standard curve is obtained by taking the potency of the lumbrokinase standard as the horizontal axis and the OD value change difference rate as the vertical axis. Figure 2 As shown, the regression equation is calculated and the regression equation is obtained: Y = 0.001662*X + 0.4127, R 2 =0.9936.

[0052] Substitute the difference rate of the OD value of the test sample into the regression equation to calculate the number of test sample potency units. Six wells should be made for each standard sample and test sample, and the average value should be used for calculation.

[0053] Test Results In the following description, the method of "WS1-(X-052)-2001Z-2010" is referred to as the original method, the method of Example 1 of the present invention is referred to as new method 1, and the method of Example 2 of the present invention is referred to as new method 2.

[0054] 1. The original method and the new method 1 were used to measure the potency of lumbrokinase (batch number: 2004001, Jiangzhong Pharmaceutical Co., Ltd.) six times to evaluate the precision of the method. The data are shown in Table 1.

[0055] Table 1 Results of six potency determinations (IU) and RSD values

[0056] 2. The original method and the new method 2 were used to measure the potency of lumbrokinase (batch number: 2004001, Jiangzhong Pharmaceutical Co., Ltd.) six times to evaluate the precision of the method. The data are shown in Table 2.

[0057] Table 2 Results of six potency determinations (IU) and RSD values

[0058] 3. The original method and the new method 1 were used to perform sample recovery determination on lumbrokinase (batch number: 2004001, Jiangzhong Pharmaceutical Co., Ltd.) with known titer to evaluate the accuracy of the method. The data are shown in Table 3.

[0059] Table 3

[0060] 4. The original method and the new method 2 were used to perform sample recovery determination on lumbrokinase (batch number: 2004001, Jiangzhong Pharmaceutical Co., Ltd.) with known titer to evaluate the accuracy of the method. The data are shown in Table 4.

[0061] Table 4

[0062] 5. The reaction time required for the new methods 1 and 2 is 5-6 h, which is 18 h compared to the original method, shortening the detection time and improving the detection efficiency.

[0063] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for determining the potency of lumbrokinase, characterized in that: The steps include: (1) Take the lumbrokinase test sample and the lumbrokinase standard, dissolve them in sodium chloride solution respectively to obtain the test sample solution and the standard solution of different concentrations; (2) Take the fibrinogen solution, add the agarose solution and the thrombin solution, mix well and pour into the ELISA plate, measure the OD value of each well with an ELISA reader, and record it as the OD blank; add the test solution and the standard solution to different wells for reaction. After the reaction is completed, measure the OD value of each well with an ELISA reader, and record it as the OD measurement; calculate the OD value change difference rate according to the following formula: ； The regression equation was calculated based on the potency of the lumbrokinase standard and the difference rate of OD value change, and the OD value of the test sample was substituted into the regression equation to calculate the potency of the test sample; The reaction time in step (2) is 5-6 hours.

2. The method according to claim 1, characterized in that: The detection wavelength of the ELISA instrument in step (2) is 490 nm.

3. The method according to claim 1, characterized in that In step (2), the concentration of the fibrinogen solution is 1.4-1.6 mg / mL, the concentration of the agarose solution is 1.4-1.6 g / 100 mL, and the concentration of the thrombin solution is 0.9-1.1 BP / mL.

4. The method according to claim 3, characterized in that In step (2), the volume ratio of the fibrinogen solution, the agarose solution and the thrombin solution is 13:13:

1.

5. The method according to claim 1, characterized in that The mass concentration of the sodium chloride solution in step (1) is 0.9%.

6. The method according to claim 1, characterized in that The concentrations of the standard solution in step (1) are 10000 IU, 9000 IU, 8000 IU, 7000 IU, 6000 IU, 5000 IU, and 4000 IU lumbrokinase units per 1 mL of the standard solution, respectively.

7. The method according to claim 1, characterized in that The temperature of the agarose solution in step (2) is 50-60°C.

8. The method according to claim 1, characterized in that: Before measuring the OD value of each well with an ELISA reader in step (2), place the plate at 20-30°C for 0.5-1 h.

9. The method according to claim 1, characterized in that: The reaction temperature in step (2) is 36-38°C.

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