Anti-platelet thrombolysin and preparation method thereof
An anti-platelet and anti-platelet aggregation technology, applied in the field of biomedicine, can solve the problems of short plasma half-life and bleeding, and achieve the effects of small bleeding tendency, high yield and obvious effect.
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Embodiment 1
[0059] Embodiment 1: Preparation of high-purity anti-platelet thrombolysin
[0060] 1. Treatment of snake venom
[0061] Accurately weigh 5 g of Agkistrodon halys venom purchased from Huizhou Venom Research Institute in Huangshan City, and dissolve it in a refrigerator at 4°C with 0.02mol / L Tris-HCl buffer solution at pH 8.0 for 6-12 hours, and then dissolve it at 4000rr / min centrifugation, centrifugation twice, each centrifugation 15min, the supernatant was collected for later use.
[0062] 2. Preliminary purification to obtain antigens for the preparation of monoclonal antibodies
[0063] (1) Anion exchange chromatography
[0064] Take the supernatant obtained from dissolving and centrifuging the crude poison in step 1 and load it on the equilibrated anion exchange chromatography DEAE column at a flow rate of 3.0ml / min. Miscellaneous proteins that cannot be adsorbed by the chromatographic medium, the elution time is 360min, and the elution flow rate is 3ml / min; then add ...
Embodiment 2
[0079] Example 2: Drug efficacy evaluation of the anti-platelet thrombolysin of the present invention
[0080] 1. Activity determination
[0081] One unit of activity of anti-thrombolysin is defined as: the amount of anti-thrombolysin that can inhibit the percentage of platelet aggregation induced by ristocetin to 100±5.
[0082] The potency determination method adopts the platelet aggregation method, as follows:
[0083] (1) Sources of platelet-rich plasma PRP and platelet-poor plasma PPP: Anticoagulant blood from blood donors with normal platelet count was taken. The anticoagulant was 3.8% sodium citrate, and the ratio of the anticoagulant to whole blood was 1:9. Centrifuge at 800r / min for 5min, absorb the supernatant to obtain platelet-rich plasma (PRP), then centrifuge at 3500r / min for 10min, absorb the supernatant to obtain platelet-poor plasma (PPP), and adjust the platelet count of PRP to (150 ~200)×10 9 .
[0084] (2) Blank tube measurement: Take a siliconized turb...
Embodiment 3
[0096] Embodiment 3: High-purity anti-platelet thrombolysin crude drug content detection method
[0097] In order to detect the content of anti-thrombolysin in serum so as to be used in pharmacokinetic tests, a set of methods for detecting the content of anti-thrombolysin raw materials by double-antibody sandwich method was established. The detection sensitivity of this method can reach 1ng / ml .
[0098] The specific detection method is as follows:
[0099] 1. Coating a monoclonal antibody: properly dilute the monoclonal antibody prepared in Example 1 of the present invention with 0.05M pH9.6 carbonate buffer, add 0.1 ml, overnight at 4°C or for 18-24 hours. The next day, the solution in the well was discarded, and washed 3 times with washing buffer for 3 minutes each time (abbreviated as washing, the same below).
[0100] 2. Adding samples: add the sample to be tested or a properly diluted standard reference product into the reaction wells, and simultaneously make blank we...
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