Universal antithrombotic substance activity detection method

Abstract

The invention discloses a universal antithrombotic substance activity detection method, and relates to the technical field of biological medicine, and the universal antithrombotic substance activity detection method comprises the following steps: diluting a to-be-detected antithrombotic substance with a 0.9% sodium chloride solution, carrying out centrifugal treatment, and sucking the supernatant as a to-be-detected solution; adding blood into a mold, standing, after the blood is coagulated into a blood clot, pressing and squeezing the blood clot into a 0.9% sodium chloride buffer solution for soaking, taking out the blood clot, and absorbing water on the surface; adding a to-be-detected solution into the centrifugal tube, and measuring an absorbance initial value of the to-be-detected solution under a preset wavelength; continuously adding the treated blood clots, shaking the centrifugal tube in a preset time, and measuring the absorbance endpoint value of the centrifugal tube under the same wavelength; and comparing the absorbance end point value and the absorbance initial value of the solution to be detected to obtain the absorbance relative variation, namely detecting the activity of the antithrombotic substance. The method has the beneficial effects that the detection method has the advantages of simplicity and convenience in operation, high cost benefit, high detection speed and the like, and is suitable for quickly screening and evaluating the activity of the antithrombotic substance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a universal method for detecting the activity of anti-thrombotic substances. Background Art

[0002] In the biomedical field, especially in antithrombotic treatment and research, accurate evaluation of the activity of antithrombotic drugs is crucial.

[0003] Traditional antithrombotic activity detection methods, such as thrombin titration, white porcelain plate titration, plasma recalcification time determination method and chromogenic substrate determination method, are relatively simple to operate and widely used in clinical and research, but there are still some limitations. The determination results are easily affected by ambient temperature and thrombin activity, which requires the experimental operation to be carried out under strictly controlled environmental conditions, and this method is only for anticoagulants that directly act on thrombin, and is not universal. The white porcelain plate titration method uses a white porcelain plate as an indicator of the titration endpoint, and the color change can be observed intuitively, which helps to determine whether the reaction is complete. The disadvantage is that the white porcelain plate titration relies on the subjective judgment of color change, which may be affected by the subjective factors of the observer, thereby affecting the accuracy of the titration. The plasma recalcification time determination method can quickly obtain results, which has its advantages for situations that require rapid diagnosis. However, this method can only be observed by the naked eye, which will cause errors, and can only be quantitatively analyzed, and qualitative analysis cannot be performed. The chromogenic substrate method has high sensitivity to the detected substances and can provide relatively accurate measurement results. However, it has the characteristics of singleness. One chromogenic substrate is only applicable to one substance. It is also costly, not widely applicable, and has certain requirements for laboratory conditions.

[0004] In recent years, with the in-depth study of antithrombotic drugs, people have begun to seek simpler, faster and cost-effective detection methods. For example, hirudin, as a potent thrombin inhibitor, the accurate determination of its anticoagulant activity is of great significance for evaluating its application prospects as a potential therapeutic agent. However, existing detection methods often cannot meet the needs of rapid and high-throughput screening, especially in drug development and preclinical research stages. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a universal method for detecting the activity of anti-thrombotic substances, which can not only overcome the limitations of the prior art, but also provide a method for detecting the activity of anti-thrombotic substances with simple operation, low cost, high efficiency, fast detection speed, wide versatility and accurate results.

[0006] The technical solution of the present invention is as follows: A universal method for detecting the activity of an antithrombotic substance comprises the following steps: The antithrombotic substance to be tested is diluted with 0.9% sodium chloride solution, then centrifuged, and the supernatant is taken as the test solution; Adding blood into the mold and letting it stand, after the blood solidifies into a blood clot, pressing and squeezing the blood clot into a container filled with 0.9% sodium chloride buffer to soak, taking out the blood clot, and absorbing surface moisture to obtain a processed blood clot; Adding the test solution into a centrifuge tube, and measuring its initial absorbance value at a preset wavelength; continuing to add the treated blood clot into the centrifuge tube, shaking the centrifuge tube for a preset time, and measuring its absorbance endpoint value at the same wavelength; The absorbance endpoint value and the absorbance initial value of the solution to be tested are compared to obtain the relative change in absorbance, that is, to detect the activity of the antithrombotic substance.

[0007] Optionally, the detection method further comprises: Add 0.9% sodium chloride buffer to another centrifuge tube as a negative control group, and measure the initial absorbance value of the negative control group at the same wavelength; continue to add the treated blood clot to the centrifuge tube, shake the centrifuge tube for a preset time, and measure the absorbance endpoint value of the negative control group at the same wavelength.

[0008] Optionally, the relative change in absorbance of the test solution and the relative change in absorbance of the negative control group are compared to detect the activity of the anti-thrombotic substance.

[0009] Optionally, the preset wavelength is 500-600 nm, and the preset time is 10-60 min.

[0010] Optionally, the preset time includes a first preset time, a second preset time and a third preset time, the first preset time is 10 minutes, the second preset time is 30 minutes, and the third preset time is 60 minutes.

[0011] Optionally, the substance to be tested is diluted 2 to 10 times with 0.9% sodium chloride solution.

[0012] Optionally, the rotation speed of the centrifugal treatment is 3000-8000 rpm, and the time of the centrifugal treatment is 2-5 minutes.

[0013] Optionally, the blood is sucked with a pipette, poured into a mold having a plurality of cylindrical grooves, and allowed to stand for 5-10 minutes; The blood is animal blood, including the blood of mice, rabbits, chickens and quails.

[0014] Optionally, after the blood coagulates into a blood clot, the blood clot is squeezed into a container containing 0.9% sodium chloride buffer and soaked for 5-10 minutes.

[0015] 10. A universal method for detecting the activity of an antithrombotic substance according to claim 1, characterized in that the antithrombotic substance to be detected comprises leech contents or leech freeze-dried powder.

[0016] The present invention has at least one of the following beneficial effects: 1. The present invention proposes a universal method for detecting the activity of antithrombotic substances, which dilutes and centrifuges the substance to be tested to remove interfering impurities, then passes the blood through a mold to coagulate it into a blood clot, ensuring the consistency of the blood clot, and removes serum factors that may affect the test results by soaking, and finally measures the change in absorbance by spectrophotometry, which can accurately evaluate the antithrombotic effect.

[0017] 2. The detection method of the present invention has the advantages of simple operation, high cost-effectiveness, fast detection speed, etc., and is suitable for rapid screening and evaluation of the activity of antithrombotic substances. The method is widely applicable and is not only suitable for the detection of leech contents, but can also be extended to the activity evaluation of other potential antithrombotic substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a physical picture of a mold used for coagulating blood into a blood clot in an embodiment of the present invention; Figure 2 This is a physical picture of a tool for turning a centrifuge tube upside down in an embodiment of the present invention. DETAILED DESCRIPTION

[0019] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0020] An embodiment of the present invention provides a universal method for detecting the activity of an antithrombotic substance, comprising the following steps: Step 1: Prepare the test substance (leech contents, leech freeze-dried powder, protein with anticoagulant activity) and dilute it 2-10 times with 0.9% sodium chloride solution. Centrifuge the diluted sample for 2-5 minutes at 3000-8000 rpm, and take the supernatant for later use. This step is to ensure that the test substance is free of impurities during spectrophotometric detection, and to physically remove possible solid impurities or cell debris to ensure the purity of the supernatant.

[0021] Step 2: Use a 160-200 μl pipette to pipette the prepared blood of experimental animals (mice, rabbits, etc.) and poultry (chickens, quails, etc.) into a Figure 1Blood clots are prepared in the cylindrical mold shown. Fresh blood is coagulated under standardized conditions to form blood clots using precise volume measurement and time control. This step ensures the consistency of the blood clots and provides a reliable sample basis for subsequent anticoagulant activity assessment.

[0022] Step 3: Let it stand for 5-10 minutes. After the blood clot solidifies, press the blood clot in the mold and squeeze it into a beaker containing 0.9% sodium chloride buffer and soak it for 5-10 minutes to gently remove the infiltrated serum components. This step not only removes serum factors that may affect the test results, but also maintains the structural integrity of the blood clot.

[0023] Step 4: Prepare three tubes of 2 ml 0.9% sodium chloride buffer as negative control and 2 ml of the substance to be tested, measure the initial value of the spectrophotometer, set the wavelength to 500-600 nm, then take out the blood clot and dry the surface water with absorbent paper, and put the blood clot into a centrifuge tube. Figure 2 The tool shown is turned upside down, and the time gradient is set to 10 minutes, 30 minutes, and 60 minutes. The spectrophotometric value of the solution is measured respectively, and the test result is accurate, the test is fast, and the applicability is wide. The present invention adopts spectrophotometry to quantitatively analyze the antithrombotic activity. By measuring the absorbance change of the solution before and after the test substance is treated, the antithrombotic effect can be accurately evaluated.

[0024] Step 5: Collect spectrophotometric data at different time points and analyze them using statistical software to create a data chart reflecting the anticoagulant effect. This chart intuitively shows the changing trend of antithrombotic activity, making it easier for experimenters to interpret the results.

[0025] like Figure 1 As shown, this embodiment provides a mold for coagulating blood into a blood clot, the mold having a plurality of cylindrical grooves, and the blood can be coagulated into a blood clot by adding blood into the grooves with a pipette and letting it stand. In this embodiment, the depth of the cylindrical groove is 3mm-7mm and the diameter is 5mm-10mm, so that the height and diameter of the blood clot prepared by the mold are consistent with the groove, that is, the height of the blood clot is 3mm-7mm and the diameter is 5mm-10mm. Through this mold, the consistency of the blood clot can be ensured, providing a reliable sample basis for the subsequent anticoagulant activity evaluation.

[0026] like Figure 2As shown, this embodiment also provides a tool for turning a centrifuge tube upside down, including a holder and a turntable; the holder is fixed on the side wall of the turntable, and the holder has a slot for fixing the centrifuge tube; the turntable is arranged along the height direction and connected to the driving device, so that it can rotate along the height direction under the drive of the driving device, thereby driving the holder to turn upside down. Since the structure and principle of the driving device of the turntable are well known to those skilled in the art, they are not described in detail in this embodiment.

[0027] The present invention discloses a universal method for detecting the activity of antithrombotic substances, which can not only overcome the limitations of the prior art, but also provide a method for detecting the activity of antithrombotic substances with the advantages of simple operation, low cost, high efficiency, fast detection speed, wide versatility and accurate results.

[0028] The present invention is further described in detail below with specific examples, but the present invention is not limited to the following specific examples.

[0029] Example 1 Leeches contain a variety of biologically active ingredients, including hirudin, which have anticoagulant and antithrombotic effects. This example aims to evaluate the antithrombotic activity of leech contents, so the contents released from leeches are directly used as test samples.

[0030] A universal method for detecting the activity of an antithrombotic substance comprises the following steps: 1) Using 30% anhydrous ethanol solution to stimulate leeches to cause them to release their contents and collect the contents in their bodies to obtain the leech contents.

[0031] 2) Dilute the prepared leech contents five times with 0.9% sodium chloride buffer, use a pipette to pipette 2 ml into a 2 ml centrifuge tube, centrifuge for 2 minutes at 5000 rpm, and remove the supernatant for later use. This step is to ensure that there is no interference from impurities when spectrophotometrically detecting the leech contents.

[0032] 3) Use a 200 μl pipette to pipette the prepared fresh quail blood into Figure 1 Blood clots were prepared in the cylindrical mold shown.

[0033] 4) Let it stand for 10 minutes. After the blood clot solidifies, press the blood clot in the mold into a beaker containing 50 ml of 0.9% sodium chloride buffer and soak for 8 minutes. Wash off the serum that has seeped out. Try to ensure that each blood clot is the same size while also maintaining the freshness of the blood clot.

[0034] 5) Prepare three centrifuge tubes with 2 ml of 0.9% sodium chloride buffer and 2 ml of leech contents, measure the initial spectrophotometric value of each tube, and set the wavelength to 510 nm; then take out the blood clots in step 4) and dry the surface moisture with absorbent paper, and put the blood clots into the above 6 centrifuge tubes. After soaking, cover the lid and place them in Figure 2 The tool shown is inverted and shaken, and the time is set to 10 minutes, 30 minutes, and 60 minutes, and the spectrophotometric value of the solution is measured respectively. By measuring the change in absorbance of the solution before and after treatment with the substance to be tested, the anti-thrombotic effect can be accurately evaluated.

[0035] 6) Create a data chart based on the measured values, see Table 1, to evaluate the anticoagulant effect.

[0036] Table 1 According to the variation trend of absorbance in Table 1, it can be seen that the coagulation activity of leech contents is significantly higher than that of sodium chloride solution.

[0037] According to the data in Table 1, it can be concluded that the leech samples have coagulation activity, and this activity increases with time.

[0038] Therefore, the present invention verifies the effectiveness and accuracy of the proposed antithrombotic substance activity detection method through the experiment of Example 1. The experimental results show that the leech contents exhibit significant antithrombotic activity, and this activity increases over time. By measuring the absorbance changes at different time points by spectrophotometry, the present method can accurately evaluate the activity of antithrombotic substances.

[0039] In addition, the detection method of the present invention has the advantages of simple operation, high cost-effectiveness, fast detection speed, etc., and is suitable for rapid screening and evaluation of the activity of antithrombotic substances. The method is widely applicable and is not only suitable for the detection of leech contents, but can also be extended to the activity evaluation of other potential antithrombotic substances.

[0040] In summary, the antithrombotic substance activity detection method of the present invention provides a new tool for the biomedical field, which helps to accelerate the development and preclinical evaluation of antithrombotic drugs.

[0041] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A universal method for detecting the activity of antithrombotic substances, characterized in that: The following steps are involved: The antithrombotic substance to be tested is diluted with 0.9% sodium chloride solution, then centrifuged, and the supernatant is taken as the test solution; Adding blood into the mold and letting it stand, after the blood solidifies into a blood clot, pressing and squeezing the blood clot into a container filled with 0.9% sodium chloride buffer to soak, taking out the blood clot, and absorbing surface moisture to obtain a processed blood clot; Adding the test solution into a centrifuge tube, and measuring its initial absorbance value at a preset wavelength; continuing to add the treated blood clot into the centrifuge tube, shaking the centrifuge tube for a preset time, and measuring its absorbance endpoint value at the same wavelength; The absorbance endpoint value and the absorbance initial value of the solution to be tested are compared to obtain the relative change in absorbance, that is, to detect the activity of the antithrombotic substance.

2. A universal method for detecting the activity of antithrombotic substances according to claim 1, characterized in that: The detection method further comprises: Add 0.9% sodium chloride buffer to another centrifuge tube as a negative control group, and measure the initial absorbance value of the negative control group at the same wavelength; continue to add the treated blood clot to the centrifuge tube, shake the centrifuge tube for a preset time, and measure the absorbance endpoint value of the negative control group at the same wavelength.

3. A universal antithrombotic substance activity detection method according to claim 2, characterized in that: The activity of the anti-thrombotic substance is detected by comparing the relative change in absorbance of the test solution with that of the negative control group.

4. A universal method for detecting the activity of antithrombotic substances according to claim 2, characterized in that: The preset wavelength is 500-600nm, and the preset time is 10-60min.

5. A universal method for detecting the activity of antithrombotic substances according to claim 4, characterized in that: The preset time includes a first preset time, a second preset time and a third preset time, the first preset time is 10 minutes, the second preset time is 30 minutes, and the third preset time is 60 minutes.

6. A universal method for detecting the activity of antithrombotic substances according to claim 1, characterized in that: The substance to be tested was diluted 2 to 10 times with 0.9% sodium chloride solution.

7. A universal method for detecting the activity of antithrombotic substances according to claim 1, characterized in that: The rotation speed of the centrifugal treatment is 3000-8000 rpm, and the time of the centrifugal treatment is 2-5 minutes.

8. A universal method for detecting the activity of antithrombotic substances according to claim 1, characterized in that: The blood is sucked with a pipette, poured into a mold having a plurality of cylindrical grooves, and allowed to stand for 5-10 minutes; The blood is animal blood, including the blood of mice, rabbits, chickens and quails.

9. A universal method for detecting the activity of antithrombotic substances according to claim 1, characterized in that: After the blood coagulates into a blood clot, the blood clot is squeezed and squeezed into a container containing 0.9% sodium chloride buffer and soaked for 5-10 minutes.

10. A universal method for detecting the activity of antithrombotic substances according to claim 1, characterized in that: The anti-thrombotic substances to be tested include leech contents and leech freeze-dried powder.

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