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Preparation method and application of anti-bungarus multicinctus toxin specific polyclonal antibody

Bungarotoxin and polyclonal antibody technology, applied in the direction of anti-animal/human immunoglobulin, material inspection products, measuring devices, etc., can solve the problems that affect the accuracy of diagnosis and the absence of bungatoxin

Pending Publication Date: 2021-11-12
SHANGHAI SERUM BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the genetic relationship between snake species and the complexity of the types and abundance of venom components, some snake venom proteins exist in various snake venoms. Once these snake venom proteins are used as detection targets, they may affect the accuracy of diagnosis due to cross-reactivity Spend
Currently, there is no fast, sensitive and specific detection reagent or product for bungarotoxin on the market

Method used

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  • Preparation method and application of anti-bungarus multicinctus toxin specific polyclonal antibody
  • Preparation method and application of anti-bungarus multicinctus toxin specific polyclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 Preparation of anti-bungarotoxin specific polyclonal antibody

[0023] 1. Antigen preparation:

[0024] Select healthy adult coral snakes, 50% male and 50% male, place the snake venom in an ice water bath immediately, and centrifuge the fresh venom at 2000g at low temperature for about 15 minutes to remove impurities. The supernatant was lyophilized into a bungatoxin dry powder for subsequent use. When using, add Tris buffer (100mM Tris, 100mM NaCl, pH8.0) to 50mg / mL snake venom concentration. The snake venom solution was mixed with an equal volume of iodoacetamide solution (200mM iodoacetamide, 100mM Tris, 100mM NaCl, pH 8.0), stirred well, and incubated at 35°C in the dark for 5 hours. After the reaction is completed, the remaining iodoacetamide is removed by ultrafiltration to obtain the bunungarotoxin antigen. The advantage of using iodoacetamide to inactivate snake venom is that the conformation of snake venom protein does not change significantly, ...

Embodiment 2

[0035] The preparation of embodiment 2bungarotoxin immunochromatography detection test strips

[0036] 1. Preparation of colloidal gold-labeled anti-bungarotoxin specific polyclonal antibody:

[0037] Heat 100mL of 0.01% chloroauric acid aqueous solution to boil, add 1.6mL of 1% trisodium citrate aqueous solution under stirring, until the color of the liquid is stable into wine red to obtain a colloidal gold solution, adjust the pH to 9.0, and add a final concentration of 30 μg / mL of anti-bungarotoxin specific monoclonal antibody, stirred for 20 minutes, added 5 mL of 10% BSA, stirred for 20 minutes, then added 1 mL of 10% PEG2000, stirred for 20 minutes, centrifuged at 5000rpm for 10 minutes, sucked out the supernatant, and Centrifuge again at 15000rpm for 30 minutes, discard the supernatant, and store the precipitate in 10 mL of 0.3% sodium tetraborate solution to obtain a colloidal gold probe solution labeled with the specific antibody of the buntoxin.

[0038] 2. Assembl...

Embodiment 3

[0040] Sensitivity and specificity test of embodiment 3bungarotoxin immunochromatography detection test strip

[0041] 1. Sensitivity detection

[0042] Bungarotoxin standard substance (bungarotoxin lyophilized powder prepared in Example 1) was serially diluted with Tris buffer (100mM Tris, 100mM NaCl, pH 8.0) (diluted concentrations were respectively 0.5ng / mL, 1ng / mL, 5ng / mL, 10ng / mL, 50ng / mL and 100ng / mL), with the test strip prepared in Example 2 for detection.

[0043] The test results showed that, when the concentration of Bungara venom was higher than 1 ng / mL, two red stripes of test line T and quality control line C appeared, and when the concentration of Bungara venom was lower than 1 ng / mL, only one red stripe of quality control line C was displayed.

[0044] 2. Specific detection

[0045] The solution (concentration is 100ng / mL, 500ng / mL, 1μg / mL etc.) of Agkistrodon halys venom of different concentrations, anti-scarve venom, cobra venom is respectively configured,...

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Abstract

The invention discloses a preparation method and application of an anti-bungarus multicinctus toxin specific polyclonal antibody. The preparation method of the anti-bungarus multicinctus toxin specific polyclonal antibody comprises the following steps: (1) preparing an antigen: extracting bungarus multicinctus toxin, and inactivating the bungarus multicinctus toxin to obtain a bungarus multicinctus toxin antigen; (2) immunizing a horse: immunizing the horse with the bungarus multicinctus toxin antigen prepared in the step (1), and collecting serum of the horse; and (3) extracting an antibody: extracting horse IgG from the serum in the step (2), and adsorbing by cobra toxin to obtain the anti-bungarus multicinctus toxin specific polyclonal antibody. According to the method, the anti-bungarus multicinctus toxin specific polyclonal antibody is prepared, and a bungarus multicinctus toxin immunochromatography detection test strip is developed on the basis of the anti-bungarus multicinctus toxin specific polyclonal antibody, and has advantages of simple operation, no complex instrument, high sensitivity, good specificity and the like so as to effectively solve the field rapid detection problem of the bungarus multicinctus toxin.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of an anti-bungarotoxin specific polyclonal antibody. Background technique [0002] Snakes are widely distributed in the world, and their species vary with regions, climates, and environments. There are snakes distributed in all provinces of our country, but most of the snake species are concentrated in the south of the Yangtze River and the southwest provinces. There are 210 species of snakes known, and 100 species are poisonous. Among them, there are 9 species of common venomous snakes on land, and marine There are 2 kinds of poisonous snakes. Among them, the corona snake is considered to be the most poisonous snake in my country. The snake has white rings and black rings arranged alternately on its back, and the white rings are narrower. This kind of snake is widely distributed in China, and it is distributed in Anhui, Zhejiang, Jiangsu, Jian...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/68G01N33/558
CPCC07K16/18G01N33/68G01N33/558
Inventor 罗敏宋立华张志凯李丹黄海斌
Owner SHANGHAI SERUM BIOTECH
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