Method for detecting activity of batroxobin

A detection method and technology of batroxobin are applied in the direction of material analysis by observing the influence of chemical indicators, and analysis by making materials undergo chemical reactions, etc., which can solve the problems of large subjective interference, high cost, and restriction of wide application. , to achieve the effect of reducing reading time, easy operation and easy control

Inactive Publication Date: 2012-11-28
ZHAOKE PHARMA HEFEI
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AI Technical Summary

Problems solved by technology

[0003] So far, there are mainly two methods for the determination of batroxobin activity: the national drug standard stipulates that the determination method of batroxobin raw material and preparation enzyme activity adopts the coagulation method, and when fibrinogen is converted into fibrin in vitro to generate a clot, Determine the titer of batroxobin according to the speed of clot formation, the sensitivity of this method can reach 2.5Bu / ml, and the reproducibility is good, but the operation of this method needs to judge the coagulation time of fibrinogen visually, and the subjective interference of the tester is relatively large The second is enzyme-linked immunosorbent assay (ELISA), its principle is to use the antigen-antibody reaction to adsorb the antigen or antibody on a solid-phase carrier, perform immunoenzyme staining on the carrier, and use a spectrophotometer to develop the color after the substrate is colored. Judgment results, based on the high catalytic efficiency of the enzyme, the reaction process can be infinitely magnified, with high sensitivity and strong specificity, but requires high-purity antibodies, which are expensive and restrict their wide application

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  • Method for detecting activity of batroxobin
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  • Method for detecting activity of batroxobin

Examples

Experimental program
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Effect test

Embodiment 1

[0027] ① Use 0.02M Tris-HCl buffer solution, the pH of the solution is 7.5, prepare 3mM S-2238 substrate solution and 16Bu / ml defibrase standard stock solution;

[0028] ②Randomly select the snake venom hemagglutinase preparation with batch number 20100409 produced by Zhaoke Pharmaceutical (Hefei) Co., Ltd. as a test sample, and the labeling amount is 1Ku / ml;

[0029] ③Add defibrase standard mother solution with activity units of 1.5, 1.2, 0.9, 0.6, 0.3, and 0.15Bu to each well of the microtiter plate, make up to 250.5 μl with 0.02M Tris-HCl buffer solution, pH=7.5, Then add 49.5 μl 3mM chromogenic substrate S-2238 solution, mix well, make 3 duplicate wells for each active unit standard product mother solution, and the reaction system is shown in Table 1;

[0030] Table 1 Defibrase standard reaction system

[0031]

[0032] ④ Add 0.2Ku snake venom hemagglutinin preparation to the wells of the microplate, volume 200μl, 49.5μl 3mM chromogenic substrate S-2238 and 50.5μl 0.02...

Embodiment 2

[0035] Select four batches of snake venom hemagglutinase preparations produced by Zhaoke Pharmaceutical (Hefei) Co., Ltd., the batch numbers are 20100315, 20100206 and 20091217, and the specifications are all 1Ku / ml. In the microtiter plate, the standard curve reaction system is the same as in Example 1, with 3 duplicate wells each. The reaction systems of the three batches of samples to be tested were 200 μl of preparation (0.2 Ku / well of enzyme), 50.5 μl of Tris-HCl solution and 49.5 μl of S-2238 substrate, and each sample had 5 duplicate wells. Put the prepared microplate plate into the microplate reader preheated for 4 minutes to 37 ° C for 15 minutes, and measure the absorbance A405 of the enzymatic hydrolysis product p-nitroaniline (PNA) in each hole at a wavelength of 405 nm in parallel to make For the standard curve of enzyme activity, the absorbance A405 value of the sample to be tested was measured, and the activity unit of the sample to be tested was calculated by s...

Embodiment 3

[0039]Take a certain batch of Batroxobin monomer with a protein content of 3.3mg / ml and 3.73mg / ml in the reserved sample after separation and purification from Zhaoke Pharmaceutical (Hefei) Co., Ltd., and use 0.02M Tris-HCl buffer solution, pH=7.5 , which was diluted to 3.3 μg / ml and 3.73 μg / ml. In the microplate plate, the standard curve reaction system was the same as that in Example 1, with 3 duplicate holes each. The reaction systems of the samples to be tested were 24 μl and 21 μl of the preparation mother solution, the amount of enzyme was 0.08 μg / well, 226.5 μl and 229.5 μl of Tris-HCl solution and 49.5 μl of S-2238 substrate, and each sample had 5 replicate wells. Immediately put the above-prepared microplate plate into a microplate reader preheated for 4 minutes to 37 ° C for 15 minutes, and then measure the absorbance A405 of the enzymatic hydrolysis product p-nitroaniline (PNA) in each hole at a wavelength of 405 nm in parallel. Make a standard curve of enzyme activ...

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Abstract

The invention discloses a method for quickly and specifically detecting the activity of batroxobin. A chromogenic substrate assay is adopted, a microplate reader is used, a linear reaction curve is obtained in a certain reaction time and a certain-activity standard substance range, a linear relation between a light absorption value of a product and the amount of the batroxobin is established, and the corresponding enzymatic activity of a sample is calculated. In addition, the recovery rate and accuracy of the standard curve are carefully researched. The method is high in accuracy, simple in steps and convenient to impellent in a laboratory, and is suitable for quality control of batroxobin raw materials and hemocoagulase preparations.

Description

technical field [0001] The invention relates to the fields of medicines and biological products, in particular to a method for measuring the activity of batroxobin by using a chromogenic substrate method. Background technique [0002] Snake venom hemagglutinin is extracted from viper venom and purified by modern biotechnology. It is an enzyme preparation mainly for hemostasis, containing two components that can promote blood coagulation, namely batroxobin and phospholipid-dependent coagulation factor X activator (FXA for short). Among them, the research on batroxobin has a history of more than 70 years. As early as 1936, Van Klobusitzky et al. purified an enzymatic hemostatic agent Batroxobin from the venom of the Brazilian lancehead snake, which is also called "batroxobin" in China. ". Batroxobin belongs to the class of serine protease molecules and is a single-chain glycoprotein that can specifically act on the Arg16-Gly17 peptide bond at the N-terminal of the Aα chain o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78
Inventor 戴向荣刘娟娟方丽杨中强李小羿张国辉
Owner ZHAOKE PHARMA HEFEI
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