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Preparation method and application of Agkistrodon acutus venom high-titer antiserum

An antiserum technology, applied in the field of immunity, can solve the problems of poor therapeutic effect, low antiserum titer, small antigenic quantity, etc., and achieve the effects of improving the therapeutic effect, stimulating the immune system, and reducing side effects.

Inactive Publication Date: 2016-10-12
WEST ANHUI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the effective way to treat Agkistrodon halys bites is to inject antiserum. The traditional method of preparing full-venom antiserum adopts the method of immunizing animals after emulsification with Freund's adjuvant. Since the hemorrhagic activity of the emulsified snake venom is still very strong, therefore, injection The amount of antigen is relatively small, otherwise the immunized animals will die, and the small amount of antigen will lead to low titer of antiserum, so the treatment effect is not good

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Step 1: Inhibition of hemorrhagic activity of Agkistrodon akistrodon

[0021] Take a clean plastic tube with a volume of 5 mL, dissolve 0.1 g of Agkistrodon acutus in 2.0 mL of 0.1M Tris-HCl (pH 8.8) buffer, add 50 mg Na 2 EDTA and fully dissolved;

[0022] Step 2: Encapsulation of snake venom protein

[0023] Add 1.4mL 30% acrylamide, 50uL 10% ammonium persulfate, 2uL tetramethylethylenediamine (TEMED) to the above solution without hemorrhagic activity, mix well, slowly add 0.5mL isopropanol along the test tube wall, 1 After 1 h, solidify, remove the isopropanol, take out the colloid and use a scalpel to score it into 4 parts, each about 1 g, and store it in a freezer;

[0024] Step 3: Inject immunized rabbits to obtain antiserum

[0025] Take 1 portion of the above colloid, add 2.0 mL of normal saline, fully grind it in a mortar, inhale it into a 5 mL disposable syringe, the total volume is about 3.5 mL, and immunize rabbits with a weight of about 2.5 kg, and injec...

Embodiment 2

[0036] Step 1: Inhibition of hemorrhagic activity of Agkistrodon akistrodon

[0037] Take a clean plastic tube with a volume of 5mL, dissolve 0.09g Agkistrodon acutus in 1.8mL 0.1M Tris-HCl (pH 8.8) buffer, add 40mgNa 2 EDTA and fully dissolved;

[0038] Step 2: Encapsulation of snake venom protein

[0039] Add 1.3mL of 30% acrylamide, 50uL of 10% ammonium persulfate, and 2uL of TEMED to the solution without hemorrhagic activity obtained in the first step, and mix well, and the other steps are the same as the second step of "Example 1";

[0040] Step 3: Inject immunized rabbits to obtain antiserum

[0041] It is the same as the third step of "Example 1".

[0042] Step 4: Determination of antiserum titer

[0043] The detection process is the same as the fourth step of "Example 1".

[0044] After calculation, the titer of the antiserum prepared in "Example 2" reached 550, while the potency of the antiserum prepared by emulsifying with conventional snake venom plus Freund's ...

Embodiment 3

[0046] Step 1: Inhibition of hemorrhagic activity of Agkistrodon akistrodon

[0047] Take a clean plastic tube with a volume of 5mL, dissolve 0.11g Agkistrodon acutus in 2.2mL 0.1M Tris-HCl (pH 8.8) buffer, add 60mgNa 2 EDTA and fully dissolved;

[0048] Step 2: Encapsulation of snake venom protein

[0049] Add 1.5mL of 30% acrylamide, 50uL of 10% ammonium persulfate, and 2uL of TEMED to the non-hemorrhagic solution obtained in the first step, and mix well, and the other steps are the same as the second step of "Example 1";

[0050] Step 3: Inject immunized rabbits to obtain antiserum

[0051] It is the same as the third step of "Example 1".

[0052] Step 4: Determination of antiserum titer

[0053] The determination of antiserum titer is similar to ELISA method. The detection process is the same as the fourth step of "Example 1".

[0054] After calculation, the titer of the antiserum prepared in "Example 3" reached 510, while the potency of the antiserum prepared by emu...

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PUM

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Abstract

The invention discloses a preparation method and an application of an Agkistrodon acutus venom high-titer antiserum. The preparation method comprises the following steps: 1, adding 50mg of Na2EDTA to every 0.1g of Agkistrodon acutus venom in order to inhibit the hemorrhage activity of Agkistodon acutus; 2, embedding the Na2EDTA processed Agkistodon acutus venom without hemorrhage activity in polyacrylamide gel; 3, grinding Agkistrodon acutus venom-containing gel, inoculating a domestic rabbit 4 times, killing the domestic rabbit in a carotid cutting manner and taking blood 3d after the fourth injection, centrifuging the blood 6h later to obtain antiserum, and cryopreserving the antiserum, wherein 1.25mL of the ground gel is hypodermically injected and 2.25mL of the ground gel is intraperitoneally injected at the first time, 3.5mL of the ground gel is intraperitoneally injected at the second, third and fourth time, and the second injection, the third injection and the fourth injection are carried out 7d after the previous injection. The preparation method guarantees the health viability of immunized animals, reduces the production cost of the antiserum and allows the high-tilter antiserum to be prepared.

Description

technical field [0001] The invention relates to a preparation method and application of a high titer antiserum of Agkistrodon akistrodon venom, belonging to the technical field of immunization. Background technique [0002] Agkistrodon ( Agkistrodon acutus ), commonly known as the five-step snake, belongs to the subfamily Viridae and the genus Agkistrodon, and is distributed in the south of the Yangtze River in my country (including Taiwan). It is a highly venomous snake unique to my country. The snake venom is a blood-circulating poisonous snake, and its main toxin is hemorrhagic toxin. The predecessors isolated 4 kinds of hemorrhagic toxins from the venom of Agkistrodon viper. After being bitten by Agkistrodon, the wound and internal organs bleed profusely, causing death and disability. high. [0003] At present, the effective way to treat Agkistrodon halys bites is to inject antiserum. The traditional method of preparing full-venom antiserum adopts the method of immun...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/06A61K39/395A61P39/02
CPCA61K39/00C07K16/06C07K16/18
Inventor 韦传宝
Owner WEST ANHUI UNIV
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