CN108220389A - The assay method of urokinase potency - Google Patents

Abstract

The present invention provides a kind of assay method of urokinase titer, comprises the following steps: step 1: the preparation of measuring material; Step 2: measure urokinase titer; 2.1, take 36 test tubes, divide into 9 rows, 4 in each row, Place in an ice bath at 2-10°C, add 0.3ml of bovine fibrinogen solution, and add 0.9, 0.8, 0.7, 0.6ml of barbiturate-sodium chloride buffer solution to each row of test tubes, and then Add 0.1, 0.2, 0.3, 0.4ml of standard solution in rows 1 to 3, add 0.1, 0.2, 0.3, 0.4ml of the first test solution in rows 4 to 6, and add 0.1, 0.2, 0.3, 0.4ml of the first test solution in rows 7 to 9 Add the second part of the test solution 0.1, 0.2, 0.3, 0.4ml respectively. Add 0.4ml of the mixed solution to the above-mentioned test tubes, shake well immediately, place in a water bath at 36.5-7-37.5°C and record the bathing time with a stopwatch, and take the time when the bubbles rise to half the volume of the reaction system as the end point of the reaction.

Description

Determination method of urokinase titer

technical field

The invention relates to a method for measuring urokinase titer.

Background technique

Urokinase (UK for short) is a kind of basic protein hydrolyzate obtained after separation and purification from healthy people and fresh human urine, which can activate plasminogen and convert it into plasmin, fibrinolytic The enzyme has a strong proteolytic ability, and the fibrinogen is converted into a fibrin clot under the action of thrombin, and the clot is hydrolyzed into a water molecule polypeptide and dissolved under the action of plasmin. Calculate the urokinase titer with the speed of dissolution of the test solution. At present, according to the urokinase variety item on page 532 of the second part of the "Chinese Pharmacopoeia" in 2015 edition, the [potency determination-bubble rise method] method is used for testing, and there are problems of weak bubbles, poor observability, and large experimental errors.

There are many methods for measuring the potency of urokinase. EP, BP, and "Chinese Pharmacopoeia" 2015 edition all adopt the bubble rising method, and only JP uses the chromogenic substrate assay method. The bubble rising method uses a lot of reagents, most of which are proteins and enzymes, which are easy to denature, and the end point of the reaction needs to be judged by the naked eye, and the operation requirements are relatively high; the chromogenic substrate method is that urokinase reacts with the substrate to generate yellow nitroaniline , measured by ultraviolet spectrophotometry, the operation is relatively simple, but reagents such as L-pyroglutamylglycyl-L-arginine-p-nitroaniline hydrochloride are difficult to purchase and expensive, and the sustainability of the method cannot Assure.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for measuring the potency of urokinase with strong observability, good test reproducibility and small experimental error.

The invention provides a kind of assay method of urokinase potency, comprises the following steps:

Step 1: Preparation of assay materials

1.1, prepare Tris buffer solution, its pH value is 9.0;

1.2, prepare barbiturate-sodium chloride buffer solution, its pH value is 7.8;

1.3. Prepare bovine fibrinogen solution;

1.4. Prepare bovine thrombin solution;

1.5. Prepare bovine plasminogen solution;

1.6. Prepare the mixed solution;

1.7. Prepare standard solution;

1.8. Prepare the test solution;

Step 2: Determination of urokinase titer

2.1. Take 36 test tubes, divide them into 9 rows, 4 tubes in each row, place them in an ice bath at 2-10°C, add 0.3ml of bovine fibrinogen solution to each row, and add barbiturate-chloride to each row of test tubes. Sodium buffer solution 0.9, 0.8, 0.7, 0.6ml, then add standard solution 0.1, 0.2, 0.3, 0.4ml in the 1st to 3rd row respectively, add the first test sample in the 4th to 6th row respectively Solution 0.1, 0.2, 0.3, 0.4ml, add the second part of the test solution 0.1, 0.2, 0.3, 0.4ml in the 7th to 9th row, respectively, add 0.4ml of the mixed solution in the above test tube, shake up immediately , put in a water bath at 36.5-7-37.5°C, and record the bathing time with a stopwatch, and take the time when the bubbles rise to half the volume of the reaction system as the end point of the reaction.

Further, the method for preparing tris buffer solution is as follows: weigh 6.06 g of tris, 3.65 g of lysine hydrochloride, 5.8 g of sodium chloride, 0.37 g of disodium edetate, Put it in a 1000ml beaker, add about 900ml of water to dissolve it, adjust the pH value to 9.0 with 0.1mol/L HCl or 0.1mol/L NaOH solution on the pH meter, finally add water to dilute to 1000ml, shake well, and store for 2- 10°C.

Further, the method for preparing the barbital-sodium chloride buffer solution is as follows: weigh 5.05 g of barbital sodium and 3.7 g of sodium chloride, put them in a 500 ml beaker, add about 400 ml of water to dissolve them, and take 0.5 g of gelatin in addition, Put it in a 50ml beaker, add about 30ml of water, heat to dissolve, add to the above solution, adjust the pH value to 7.8 with 0.2mol/L HCl solution on the acidity meter, finally add water to dilute to 500ml, shake well, and store for 2- 10°C.

Further, the method for preparing bovine fibrinogen solution is as follows: take fibrinogen (bovine blood), add the barbiturate-sodium chloride buffer solution prepared in step 2, and make a solution containing 6.67 mg of coagulable protein per 1 ml, Shake well, that is, set at 2-10 ℃.

Further, the method for preparing bovine thrombin solution is as follows: take thrombin (cow blood), add the barbiturate-sodium chloride buffer solution prepared in step 2, make a solution containing 6.0 units of thrombin per 1 ml, place in 2- 10°C.

Further, the method for preparing bovine plasminogen solution is as follows: take plasminogen (bovine blood), add the Tris buffer prepared in step 1, and make every 1ml contain 0.7-1.4 casein Unit solution, set at 2-10°C. If the resulting solution is turbid, centrifuge and take the supernatant for use.

Further, the method for preparing the mixed solution is as follows: Take bovine thrombin solution and bovine plasminogen solution and mix them evenly in equal volumes, and place at 2-10°C.

Further, the method for preparing the standard solution is as follows: take 1 standard urokinase, add the barbiturate-sodium chloride buffer solution prepared in step 2, dissolve and dilute it into a solution containing about 60 units of urokinase per 1 ml , set at 2-10°C.

Further, prepare the test solution: take 5 bottles of the test product, put them in a volumetric flask, add the barbiturate-sodium chloride buffer solution prepared in step 2 to dissolve, mix, and dilute to the same concentration as the standard solution , set at 2-10°C.

Further, take the logarithm value of the concentration of each tube of the standard solution as the abscissa, and the logarithm value of the difference between the reaction end time and the bathing time as the ordinate, calculate the linear regression equation, and then take the reaction end time of each tube of the test product and the bathing time as the ordinate. Input the logarithmic value of the time difference into the regression equation, calculate the number of units in each tube, divide by the sample volume of each tube, that is, the number of units per 1ml, find the average value, and calculate the number of titer units according to the following formula:

By adopting the method for measuring urokinase titer provided by the present invention, the bubbles generated during the test of urokinase titer are large and bright, the reaction end point is obvious, the observability is strong, the test reproducibility is good, and the experimental error is small.

Description of drawings

The accompanying drawings described here are used to provide a further understanding of the present invention and constitute a part of the application. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations to the present invention. In the attached picture:

Fig. 1 schematically shows the linear diagram of the standard product of Example 3 of the present invention.

Detailed ways

Example 1

The embodiment of the present invention provides a method for determining the titer of urokinase, comprising the following steps:

Step 1: Preparation of assay materials

1.1, prepare Tris buffer solution (its pH value is 9.0)

Weigh 6.06g of trishydroxymethylaminomethane, 3.65g of lysine hydrochloride, 5.8g of sodium chloride, and 0.37g of disodium ethylenediaminetetraacetate, put them in a 1000ml beaker, add about 900ml of water to dissolve them, and put them on the pH meter Use 0.1mol/L HCl or 0.1mol/L NaOH solution to adjust the pH value to 9.0, and finally add water to dilute to 1000ml, shake well, and store at 2-10°C.

1.2, prepare barbiturate-sodium chloride buffer solution (its pH value is 7.8)

Weigh 5.05g of barbital sodium and 3.7g of sodium chloride, put them in a 500ml beaker, add about 400ml of water to dissolve them, take another 0.5g of gelatin, put them in a 50ml beaker, add about 30ml of water, heat to dissolve and incorporate into the above solution , adjust the pH value to 7.8 with 0.2mol/L HCl solution on the acidity meter, and finally add water to dilute to 500ml, shake well, and store at 2-10°C.

1.3. Preparation of bovine fibrinogen solution

Take fibrinogen (bovine blood), add the barbiturate-sodium chloride buffer solution prepared in step 2 according to the amount indicated on the label, and make a solution containing 6.67 mg of coagulable protein per 1 ml, shake well, and obtain, Set at 2-10°C.

1.4. Preparation of bovine thrombin solution

Take thrombin (cow blood), add the barbiturate-sodium chloride buffer solution prepared in step 2 according to the amount indicated on the label, to make a solution containing 6.0 units of thrombin per 1 ml, and place at 2-10°C.

1.5. Prepare bovine plasminogen solution

Take plasminogen (bovine blood), add the tris buffer solution prepared in step 1 according to the amount indicated on the label, and make a solution containing 0.7-1.4 casein units per 1ml, and place in 2-10 ℃. If the resulting solution is turbid, centrifuge and take the supernatant for use.

1.6. Prepare the mixed solution

Before use, take the bovine thrombin solution prepared in step 4 and the bovine plasminogen solution prepared in step 5 and mix them evenly in equal volumes to obtain the product, and place it at 2-10°C.

1.7. Preparation of standard solution

Take 1 urokinase standard product, add the barbiturate-sodium chloride buffer solution prepared in step 2, dissolve and dilute to a solution containing about 60 units of urokinase per 1 ml, and place at 2-10°C.

1.8. Prepare the test solution

Take 5 bottles of the test product, put them in a volumetric flask, add the barbiturate-sodium chloride buffer solution prepared in step 2 to dissolve, mix evenly, and dilute to the same concentration as the standard solution, and place at 2-10°C.

Above, gelatin, standard urokinase, fibrinogen (bovine blood), thrombin (bovine blood), and plasminogen (bovine blood) were provided by China National Institute for the Control of Pharmaceutical and Biological Products, and urokinase was provided by domestic Products of raw material and preparation manufacturers.

Step 2: Determination of urokinase titer

2.1. Take 36 test tubes (12×100mm), divide them into 9 rows, 4 tubes in each row, place them in an ice bath at 2-10°C, add 0.3ml of bovine fibrinogen solution to each row, and add barium to each row of test tubes. 0.9, 0.8, 0.7, 0.6ml of Bito-sodium chloride buffer solution, then add standard solution 0.1, 0.2, 0.3, 0.4ml in the 1st to 3rd row respectively, and add the 4th to 6th row respectively Add 0.1, 0.2, 0.3, 0.4ml of 1 portion of test solution, and add 0.1, 0.2, 0.3, 0.4ml of 2nd portion of test solution in the 7th to 9th row respectively. Add 0.4ml of the mixture to the above test tubes, shake well immediately, place in a water bath at 36.5-7-37.5°C, and record the bathing time with a stopwatch, and take the time when the bubbles rise to half the volume of the reaction system as the end point of the reaction.

2.2. Take the logarithm value of the concentration of each tube of the standard solution as the abscissa, and the logarithm value of the difference between the reaction end time and the bathing time as the ordinate, calculate the linear regression equation, and then use the reaction end time and bathing time of each tube of the test product The logarithmic value of the difference is input into the regression equation, the number of units in each tube is calculated, divided by the sample volume of each tube, the number of units per 1ml is obtained, the average value is obtained, and the number of titer units is calculated according to the following formula:

Embodiment 2 Orthogonal Test

1. Formulate an orthogonal experiment analysis table, select the influence of the reagent placement temperature and reaction temperature on the bubble generation to do an orthogonal experiment, and select 3 levels for each factor.

Starting from the addition of bovine fibrinogen (F) solution, the temperature and reaction temperature after the addition of reagents are selected to be different, and an orthogonal experimental analysis table is formulated. See Table 1 and Table 2.

Table 1 Orthogonal experiment table

Table 2 Orthogonal experiment table

1. Select the effect of adding reagent storage temperature and reaction temperature on the amount of air bubbles to conduct an orthogonal experiment, select 3 levels for each factor, and draw a 2-factor 3-level orthogonal test table. Take 4 small test tubes each time, add 0.3ml of bovine fibrinogen solution to each, put them in a water bath at the temperature listed in the "adding reagent storage temperature" corresponding to the serial number 1-9 in the orthogonal experiment table, and then add barbiturate respectively -sodium chloride buffer solution and need testing solution, add mixed solution (bovine thrombin solution and bovine plasminogen solution) at last, shake up, put into the "reaction temperature" corresponding to sequence number 1-9 in the orthogonal experiment table Carry out the reaction at the listed temperature, record the time of bathing with a stopwatch, and observe the amount and rise of small bubbles in the clot in the test tube at the same time. When the bubbles rise to half the volume of the reaction system as the end of the reaction, time it immediately.

Table 3 Orthogonal experiment results

It can be seen from the orthogonal results that the significant order of the influence of the two factors on the amount of bubbles produced during the determination of the urokinase titer is: adding the reagent and placing it >reaction temperature, and the best experimental conditions are: adding the reagent and placing it the most The optimum temperature is 2-10°C, and the optimum reaction temperature is 36.5-7-37.5°C.

Embodiment 3 repeatability test

The water bath mode after the modification of Example 2 was used to carry out 6 experiments on 9 batches of samples at different time periods. The correlation of the standard items was all greater than 0.9997, and the maximum RSD of the samples was 1.6%, and the minimum was 0.5%. Refer to Figure 1.

Table 4 urokinase titer repeatability results for injection

As can be seen from the above description, according to the method for measuring the urokinase titer in Example 1, the bubbles produced in the urokinase titer test process are large, bright, and the reaction end point is obvious, the observability is strong, and the test reproducibility is good. The experimental error is small.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. the assay method of urokinase titer is characterized in that, comprises the following steps: Step 1: Preparation of assay materials 1.1, prepare Tris buffer solution, its pH value is 9.0; 1.2, prepare barbiturate-sodium chloride buffer solution, its pH value is 7.8; 1.3. Prepare bovine fibrinogen solution; 1.4. Prepare bovine thrombin solution; 1.5. Prepare bovine plasminogen solution; 1.6. Prepare the mixed solution; 1.7. Prepare standard solution; 1.8. Prepare the test solution; Step 2: Determination of urokinase titer Take 36 test tubes, divide them into 9 rows, 4 tubes in each row, place them in an ice bath at 2-10°C, add 0.3ml of bovine fibrinogen solution to each row, and add barbiturate-sodium chloride buffer to each row of test tubes solution 0.9, 0.8, 0.7, 0.6ml, and then add the standard solution 0.1, 0.2, 0.3, 0.4ml in the 1st to 3rd row, respectively, add the first part of the test solution 0.1ml in the 4th to 6th row , 0.2, 0.3, 0.4ml, add the second part of the test solution 0.1, 0.2, 0.3, 0.4ml respectively in the 7th to 9th rows; Add 0.4ml of the mixed solution to the above test tubes, shake immediately, place in a water bath at 36.5-7-37.5°C, and record the bathing time with a stopwatch, and take the time when the bubbles rise to half the volume of the reaction system as the end point of the reaction. 2. the assay method of urokinase titer as claimed in claim 1 is characterized in that, the method for preparing tris buffer is as follows: take tris 6.06g, lysine hydrochloride 3.65 g, 5.8g of sodium chloride, 0.37g of disodium edetate, put in a 1000ml beaker, add about 900ml of water to dissolve it, and adjust the pH value to 9.0, finally add water to dilute to 1000ml, shake well, and store at 2-10°C. 3. the assay method of urokinase titer as claimed in claim 1 is characterized in that, the method for preparing barbital-sodium chloride damping fluid is as follows: take barbital sodium 5.05g, sodium-chlor 3.7g, Put it in a 500ml beaker, add about 400ml of water to dissolve it, take another 0.5g of gelatin, put it in a 50ml beaker, add about 30ml of water, heat it to dissolve, add it to the above solution, and use 0.2mol/L HCl on the pH meter to adjust the pH value to 7.8, finally add water to dilute to 500ml, shake well, and store at 2-10°C. 4. the assay method of urokinase titer as claimed in claim 1 is characterized in that, the method for preparing bovine fibrinogen solution is as follows: get fibrinogen (bovine blood), add the barbiturate-chloride of step 2 preparation Sodium chloride buffer solution is made into a solution containing 6.67 mg of coagulable protein per 1 ml, shaken well, and placed at 2-10°C. 5. the assay method of urokinase titer as claimed in claim 1 is characterized in that, the method for preparing bovine thrombin solution is as follows: get thrombin (cow blood), add the barbiturate-sodium chloride of step 2 preparation The buffer solution is made into a solution containing 6.0 units of thrombin per 1ml, and placed at 2-10°C. 6. the assay method of urokinase titer as claimed in claim 1 is characterized in that, the method for preparing bovine plasminogen solution is as follows: get plasminogen (bovine blood), add step 1 preparation three Hydroxymethylaminomethane buffer solution, made into a solution containing 0.7-1.4 casein units per 1ml, placed at 2-10°C, if the prepared solution is turbid, centrifuge and take the supernatant for use. 7. the assay method of urokinase titer as claimed in claim 1 is characterized in that, the method for preparing mixed solution is as follows: get bovine thrombin solution and bovine plasminogen solution equivolume mix, put 2-10 ℃. 8. the assay method of urokinase titer as claimed in claim 1 is characterized in that, the method for preparing standard solution is as follows: get 1 urokinase standard product, add the barbiturate-sodium chloride buffer prepared in step 2 solution, dissolved and diluted to a solution containing about 60 units of urokinase per 1ml, and placed at 2-10°C. 9. the assay method of urokinase titer as claimed in claim 1 is characterized in that, preparation need testing solution: get 5 bottles of need testing, put in volumetric flask, add the barbiturate-chlorination of step 2 preparations Sodium buffer solution is dissolved, mixed evenly, and diluted to the same concentration as the standard solution, and placed at 2-10°C. 10. the assay method of urokinase titer as claimed in claim 1 is characterized in that, the logarithmic value of each tube concentration with standard solution is abscissa, and the difference logarithmic value of reaction terminal time and bathing time is ordinate , calculate the linear regression equation, and then input the logarithmic value of the difference between the reaction end time of each tube of the test product and the bathing time into the regression equation, calculate the number of units in each tube, divide it by the sample volume of each tube, and get the The number of units, the average value was calculated, and the number of potency units was calculated according to the following formula: