Application of procyanidins and fibrinogen determination reagents containing procyanidins
A fibrinogen and proanthocyanidin technology, applied in biological testing, material testing, etc., can solve problems such as unsatisfactory thrombin stabilization effect, and achieve the effect of improving stability and reducing catalytic effect.
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Embodiment 1
[0026] Effect of proanthocyanidins on coagulation reaction of thrombin
[0027] Prepare 50 mM Tris, in which calcium chloride is 10 mM, and the concentration of fibrinogen is 2.5 mg / ml. In the microwell of a 96-well microplate reader, add the above-mentioned Tris solution containing calcium chloride and fibrinogen. At the same time, 200 μL of proanthocyanidin-free thrombin (control sample) and 200 μL of proanthocyanidin-containing thrombin were added to corresponding microwells. In the thrombin containing proanthocyanidins, the concentrations of proanthocyanidins are respectively 0.05%, 0.1%, 0.5%, 1%, and 1.5% (mass volume percentage). Absorbance values at 595 nm were measured. The measurement interval is 10s, the time is 15min, and the measurement temperature is 37°C. In order to determine the maximum blood coagulation reaction rate V on each absorption light curve under different conditions max (△m,OD / min), the result is as follows figure 1 Shown: when proanthocyanid...
Embodiment 2
[0030] Analysis of Thrombin Inhibition Parameters
[0031] Prepare 50mM Tris, add thrombin dry powder therein, and prepare the thrombin solution with a thrombin concentration of 50U / mL as a control sample; in the above thrombin solution, add 1.5% proanthocyanidins (mass volume ratio) to prepare A mixed system of thrombin and proanthocyanidins. After 10 minutes, 200 μL of the above-mentioned two groups of prepared components were respectively taken and added to the reaction wells of a 96-well microplate reader. The reaction wells contained 30 μL of the luminescent substrate D-Phe-Pip-Arg-pNA, and the concentrations of the luminescent substrates were 1 mM, 1.5 mM, 2 mM, 3 mM and 5 mM, respectively. Every 10s, the measurement time is 10min. The reaction speed is expressed by the increment of pNA per unit time, the unit is △μmol / min, and the extinction coefficient ε of pNA is 8270 / M / cm. Draw the Lineweaver-Burk curve of the control sample and the sample containing 1.5% proantho...
Embodiment 3
[0035] Prepare fibrin assay reagents, and study the effect of proanthocyanidins on the results of fibrinogen assay.
[0036] First prepare 50mM Tris buffer, add proanthocyanidin 1.0%, α-alanine 1.5%, calcium chloride 9mM, polyethylene glycol 0.1%, mannitol 2.3%, maltose 1.6%, potassium sorbate 1%. After mixing evenly, add thrombin freeze-dried powder to prepare a solution with a final thrombin concentration of 100 U / mL to obtain a liquid fibrinogen assay reagent.
[0037] Add 0.1%, 0.5%, 1%, and 1.5% proanthocyanidins to the prepared liquid fibrinogen assay reagents to prepare fibrinogen assay reagents with different proanthocyanidin contents.
[0038] Prepare the buffer solution for FIB-rated plasma: weigh 2.5g of barbiturate, 2.75g of barbital sodium, and 7.3g of sodium chloride, dissolve them in 750mL of deionized water, use 0.1mol / L hydrochloric acid to adjust the pH to 7.35, add water to 1L.
[0039] Reconstitute fibrinogen plasma with 1mL distilled water, dilute it wi...
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