Rosmarinic acid-containing thromboplastin time measuring reagent
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A technology of thrombin time and rosmarinic acid, which is applied in the field of thrombin time determination reagents and can solve the problems of high toxicity and the like
Active Publication Date: 2016-03-23
QINGDAO GUGAO BIOTECH CO LTD
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Sodium azide has good antibacterial properties, but it is more toxic
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Embodiment 1
[0033] Influence of Rosmarinic Acid on Antioxidation of TT Determination Reagent
[0034] Antioxidation Test of Thrombin Time Determination Reagent Containing Rosmarinic Acid at Room Temperature
[0035] Prepare a buffer solution with a Tris concentration of 50 mM (pH7.4), and add a final concentration of 2% BSA, 0.05% sodium azide, 3.5% methionine, 1.5% PEG, 3% Mannitol, 2.5% maltose, and 10 mM calcium chloride, all of which except calcium chloride are mass volume fractions. The above mixed solution was filtered with a 0.22 micron filter membrane, and a freeze-dried preparation of human thrombin was added to the filtrate so that the concentration of human thrombin in the final filtrate was 25 U / ml to obtain a TT assay reagent. The TT value of the reagent was determined by CA1500 coagulation analyzer to be 15.8s.
[0036] Contrast reagents: Studies have shown that in blood coagulation preparations containing thrombin, the addition of high concentrations of rosmarinic acid no...
Embodiment 2
[0043] Long-term Antioxidative Test of Thrombin Time Determination Reagent Containing Rosmarinic Acid
[0044] Divide the TT reagent without rosmarinic acid and the TT reagent containing rosmarinic acid into separate packages, store at 4°C, take out one bottle every month, and take 9 consecutive months to detect the TT of the two reagents. value.
[0045] Regression equation:
[0046] 3.5% methionine: y=0.0352x+15.766; CV=4.2%
[0047] 3.5% methionine and 1.5mM rosmarinic acid: y=0.0187x+15.726; CV=3.5%
[0048] This shows that in the above time period, due to the addition of rosmarinic acid, the antioxidant performance of TT reagent was significantly improved.
[0049] The rosmarinic acid mentioned in the present invention has potential biological activity, including its antioxidant properties. Adding rosmarinic acid to preparations containing thrombin can inhibit the activity of thrombin to a certain extent, but it will not affect the coagulation mechanism under the ...
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Abstract
The invention relates to a rosmarinic acid-containing thromboplastin time measuring reagent. The rosmarinic acid-containing thromboplastin time measuring reagent is composed of a Tris-HCl buffer containing 25 U / ml of human thrombin, 2% of bovine serum albumin, 1.5 mM of rosmarinic acid, 0.05% of sodium azide, 3.5% of methionine, 1.5% of PEG, 3% of mannitol, 2.5% of maltose and 10 mM of calcium chloride, wherein percentage is mass-volume ratio, and a pH value is 7.4. A preparation method of the reagent comprises the following steps: preparing the buffer with the pH value being 7.4 and Tris concentration being 50 mM, adding BSA with final concentration being 2%, 1.5mM of rosmarinic acid with final concentration being 2%, triazacyclononane with final concentration being 0.05%, methionine with final concentration being 3.5%, PEG with final concentration being 1.5%, mannitol with final concentration being 3%, maltose with final concentration being 2.5%, and 10mM of calcium chloride in the buffer, wherein, besides calcium chloride, the unit of the materials is mass-volume fraction; filtering a mixed liquor with a 0.22-micrometer filter membrane, and adding a human thrombin freeze-drying preparation in a filtrate so that the concentration of human thrombin in a final filtrate is 25 U / ml, and obtaining a TT reagent. According to the invention, rosmarinic acid is taken as a stabilizing agent, so that antioxidation performance and stabilization performance of the thromboplastin time measuring reagent can be obviously increased.
Description
[0001] This application is a divisional application with application number 2015104204159, the application date is 2015-07-16, and the application name is "an antioxidant for blood coagulation reagent". technical field [0002] The invention relates to a reagent for measuring thrombin time containing rosmarinic acid. Background technique [0003] Coagulation tests belong to the examination of thrombotic diseases, among which thrombin time (TT) is an important indicator of coagulation function examination, which is used to examine the ability of plasma fibrinogen to convert into fibrin. [0004] The core component of the thrombin time assay reagent is thrombin. Thrombin is a multifunctional serine protease with a similar sequence and structure to chymotrypsin. It contains two polypeptide chains, A and B, connected by an interchain disulfide bond. The B chain is a functional chain with a typical fold structure of serine proteolytic enzymes, containing an active center located...
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