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Preparation and use method of antibody resisting scorpion venom of buthus martensii karsch in China F (ab') 2

A technology of East Asian scorpion venom and antibody, which is applied in the preparation of scorpion venom antibody, anti-Chinese East Asian scorpion venom F2 antibody and preparation technology, which can solve the adverse reactions of increased drug usage, low purity, and increased drug consumption and other problems, to achieve the effects of reducing the incidence of adverse reactions, improving enzyme digestion efficiency, and shortening the production cycle

Inactive Publication Date: 2014-06-18
中国人民解放军成都军区疾病预防控制中心
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AI Technical Summary

Problems solved by technology

The current general preparation technology for the production of scorpion venom antiserum has the following defects: the immune plasma is first enzymatically hydrolyzed, and due to the non-specificity of the pepsin action site, other proteins (such as albumin, fibrinogen, etc.) , lipoprotein, etc.) are also hydrolyzed, which brings great difficulties for later purification
At the same time, the amount of ammonium sulfate used in the preparation process is relatively large, and the production cycle is long.
If the caprylic acid precipitation method is used for purification, the production cost will double due to the high price of caprylic acid (above 500 yuan / L), and because caprylic acid has a mild stimulating effect on the eyes, skin and mucous membranes, inhalation of steam will cause coughing
The thermal decomposition produces corrosive and irritating fumes, is harmful to the environment, and can cause pollution to water bodies, so it cannot be used in the production of pharmaceuticals.
[0011] Secondly, the existing process does not take further purification measures except salting out and heating. Such a process leads to more unenzymolyzed IgG and enzymolyzed Fc fragments in the prepared product, and the purity of the product can only reach about 70%. , which can easily lead to the occurrence of allergic reactions in clinical application
At the same time, the potency of the product is reduced due to the low purity, and it is necessary to increase the dosage of the drug to achieve the treatment effect, and the increase of the dosage of the drug increases the occurrence of adverse reactions.

Method used

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  • Preparation and use method of antibody resisting scorpion venom of buthus martensii karsch in China F (ab') 2
  • Preparation and use method of antibody resisting scorpion venom of buthus martensii karsch in China F (ab') 2
  • Preparation and use method of antibody resisting scorpion venom of buthus martensii karsch in China F (ab') 2

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Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1, the preparation of antibody

[0049] East Asian scorpion venom was purchased from ground scorpion farms in Jiangxi, Shandong, and Henan. The LD50 in the abdominal cavity was measured and used as an immunogen. The LD50 of the intraperitoneal injection of East Asian scorpion venom in mice: produced in Shanxi was 1.4-2.0 mg·kg-1 ﹒ Produced in Shandong is 2.0~3.0mg·kg-1. 2.0-3.5mg kg-1 produced in Henan Province; Kunming pony, 4-15 years old, qualified for quarantine according to the relevant provisions of the three parts of the "Chinese Pharmacopoeia" in 2010, Kunming white mice (clean grade, body weight 18 ~22g,), pepsin passed the test of class A substances, and the specific activity was 1:3000.

[0050] (1) Immunization and immune plasma collection

[0051] Weigh the appropriate amount of BMK scorpion venom produced in Shanxi, Shandong and Henan according to the ratio of 3:1:1, and dissolve 0.9% sodium chloride injection to prepare 10 mg·mL -1 The solu...

Embodiment 2

[0062] Anti-East Asian scorpion IgG and F(ab') 2 Antibody Titer Detection

[0063] (1) Determination of antibody titer by ELISA method

[0064] Coating solution (0.05mol﹐L -1 Carbonate buffer, pH 9.6) to dilute scorpion venom to a final protein concentration of 0.5 mg. mL -1 After coating the ELISA plate, 100 μL per well, washed the plate 3 times with washing solution after staying overnight at 4°C. Add 100 μL of 10% calf serum solution to each well, and block for 1 h at 37 °C. The sample to be tested was carried out with diluent (PBS buffer solution containing 10% calf serum, pH7.4) for 10 1 ~10 10 The 10-fold serial dilution of the serum was used as a negative control, and each sample was added with 3 duplicate wells, and the plate was washed at 37°C for 2 hours. Add 100 μL of HRP-labeled secondary antibody (rabbit anti-horse), bind at 37°C for 2 hours, and then wash the plate. Add the newly prepared o-phenylenediamine (OPD) color development solution, and then add H...

Embodiment 3

[0073] Preparation of Antibody Preparations

[0074] Weigh 24g of refined anti-East Asian scorpion venom F(ab') 2 Antibody freeze-dried product, reconstituted with 5050ml of water for injection, filtered through a 0.22um membrane, subpackaged, 5ml / bottle, and freeze-dried.

[0075] Finally prepared freeze-dried anti-East Asian scorpion scorpion venom F(ab') 2 The finished antibody preparation was tested by the mouse neutralization method. Each bottle can effectively neutralize the average LD50 of 11.9mg of East Asian pincer scorpion venom produced in Shanxi to a 70kg adult, and can neutralize 24mg of BMK scorpion venom produced in Shandong and Henan, and BMK scorpion produced in Liaoning. Poison 48mg. It can effectively neutralize the lethal dose of scorpion venom entering the human body.

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Abstract

The invention relates to a preparation and use method of an antibody resisting scorpion venom of buthus martensii karsch (BMK) in China F (ab') 2. The antibody enables mouse survival rate to reach 100% when ratio of the antibody to scorpion venom of BMK in Shanxi is 2: 1 (M: M), enables mouse survival rate to reach 100% when the ratio of the antibody to scorpion venom of BMK in Liaoning is 0.5: 1 (M: M), and also enables the mouse survival rate to reach 100% when the ratio of the antibody to scorpion venom of BMK in Shandong and Henan is 1: 1 (M: M); and the immunodiffusion detection result shows that a clear immunoprecipitation line appears when the mass ratio of the antibody to scorpion venom of BMK from various regions reaches 0.4: 1. According to the preparation method, a horse is immunized with scorpion venom of buthus martensii kirsch to gain immunized blood plasma, and the immunized blood plasma is subjected to salting out for preparing IgG (Intravenous Gamma Globulin), enzymolysis and purification by an ion exchange column, so as to obtain a high-purity F (ab') 2 active fragment.

Description

technical field [0001] The invention relates to the preparation of a scorpion venom antibody, which belongs to the field of biomedical products, in particular to an anti-Chinese East Asian scorpion scorpion venom F(ab')2 antibody and a preparation process. technical background [0002] Scorpions belong to Phylum Arthropoda, Order Scorpionidae, including more than 800 species of scorpions. According to reports, there are nearly 50 species of scorpions that are harmful to humans and have medical significance, and almost all of them belong to the scorpion family. There are 29 species of scorpions in China, and there are about ten species of scorpions in China, among which the most widely distributed is the East Asian scorpion (Buthus martensii Karsch, BMK). The East Asian scorpion, also known as the Martensii scorpion, is mainly distributed in Liaoning, Inner Mongolia, Shandong, Shanxi, Shaanxi, Hebei, Henan, Hubei, Hunan, Anhui, Jiangsu and other provinces in China. The toxi...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/06C12P21/06C07K1/36C07K1/34C07K1/16
Inventor 郑颖范泉水邱薇张富强
Owner 中国人民解放军成都军区疾病预防控制中心
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