Preparation of egg antigen for detecting Ornithobilharziasis and colloidal gold labeled detection test strip

A technology for detection of oriental flukes and test strips, which is applied in the field of colloidal gold test strips for the purification of crude antigens of oriental fluke eggs, purified antigens, and detection of schistosomiasis antibodies, which can solve the problems of missed detection rate, low detection rate, and long time-consuming pathogen detection and other problems, to achieve the effect of high purity of eggs

Pending Publication Date: 2019-12-10
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pathogen inspection takes a long time and the detection rate is low, resulting in a certain missed detection rate, which affects the control of the disease

Method used

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  • Preparation of egg antigen for detecting Ornithobilharziasis and colloidal gold labeled detection test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The acquisition of the worm body and ovum of oriental fluke of embodiment 1

[0020] In the oriental birch disease endemic area of ​​Tibet, the positive sheep infected by oriental birch were obtained by fecal examination. The detection method is carried out by Zhang Shiying's improved manure incubation method, specifically: take 20 grams of sheep manure, add water to make it thin, and sieve it in sequence. The first layer is 40 holes / inch "copper sieve", and the second layer is 260 holes / inch." Nylon sieve”, and then wash the nylon sieve in water for 3 to 5 times (a bucket of water can wash dozens of feces), add the feces in the sieve to a type III flask (250ML capacity), add water to the bottle mouth, Add a ball of cotton to the neck of the bottle, pour off the mixed water on the cotton, add clear water and incubate in an incubator at 20°C-30°C, observe the hatching of miracidia every other hour, observe three times in total, and judge positive if there are miracidia. ...

Embodiment 2

[0023] Example 2 Extraction of worm egg soluble antigen:

[0024] Take the eggs obtained above and place them in a grinder, add physiological saline at a volume ratio of 1:1-5 (the most suitable is 1:3), crush them in the grinder, and no complete eggs are found under the microscope; -40°C to 10°C Repeated freezing and thawing 3 times, ultrasonication on ice (ultrasonic intensity 100-1000w, 900w is the most suitable), ultrasonic 2 seconds interval 9 seconds, a total of 0.5-2 hours (1 hour is the most suitable), after ultrasonication, centrifuge at 12000r / min for 5 minutes, take The soluble supernatant, which is the crude extract of soluble antigen of eggs, was frozen at -20°C. Before production, centrifuge the egg soluble antigen again at 12,000r / min for 5 minutes, heat the supernatant to 100°C and keep it for 5-30 minutes to denature part of the protein, and centrifuge at 12,000r / min for 5-30 minutes to remove denatured protein and other precipitates. The above obtained is th...

Embodiment 3

[0025] The preparation of embodiment 3 colloidal gold immune test strips

[0026] 1) The preparation of the soluble antigen of insect eggs for detection is as described in Example 2.

[0027] 2) Spotting on nitrocellulose membrane: Dilute the soluble antigen of Orientobilax worm eggs to 0.5 mg / ml for marking the detection line (T line), and dilute the mouse anti-His tag monoclonal antibody to 0.5 mg / ml is used for the streaking of the quality control line (C line). Stick the NC film on the bottom plate, and then use the colloidal gold spotting system to draw the line. The amount of the line is 1µl / cm. Desiccant, valid for 15 months.

[0028] 3) Preparation of colloidal gold: Colloidal gold is fired according to sodium citrate reduction method: All glassware for firing and storing colloidal gold should be clean. Take the dried Erlenmeyer flask and add deionized water and chloroauric acid with a final concentration of 0.01%, and heat to boiling. Quickly add 1.5 times the vo...

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Abstract

The invention relates to preparation of an egg antigen for detecting Ornithobilharziasis and a colloidal gold labeled detection test strip. The Ornithobilharziasis antibody detection colloidal goldimmunoassay reagent strip is provided with a sample suction pad, a gold labeled pad, an NC membrane, an absorption pad, a PVC bottom plate, a detection line (T line) and a control line (C line), and the nitrocellulose membrane of the colloidal gold labeled detection test strip is coated with the Ornithobilharziasis egg purified antigen as a detection substance. The detection reagent strip provided by the invention is accurate and reliable in detection effect, simple and easy to operate and low in cost, is more suitable for field epidemiological investigation, disease screening and immunological diagnosis of low epidemic areas of Ornithobilharziasis , and is suitable for large-scale popularization and application.

Description

technical field [0001] The invention relates to the technical field of protein purification and detection, in particular to the technical field of egg antigen purification and antibody detection for oriental fluke detection, and specifically refers to a method for purifying crude antigen of oriental fluke egg, related purified antigens and Schistosoma antibody detection colloidal gold test strips. Background technique [0002] Orient birchasiasis is a kind of schistosomiasis caused by Orient birch parasitizing in the portal vein and mesenteric vein of the host. Worldwide distribution. India, Mongolia, Iraq, Kazakhstan in Asia and Russia, France and other countries in Europe all have the occurrence of this disease. In my country, oriental fluke disease is mainly distributed in Gansu, Xinjiang, Sichuan, Guizhou, Inner Mongolia, Heilongjiang and other vast areas. [0003] Oristania is subdivided into Ornsima pensdorf, A. schematica, A. turkestan and A. nodularis var. turkest...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/532G01N33/558G01N33/58G01N33/68
CPCG01N33/532G01N33/587G01N33/6854G01N33/558
Inventor 朱传刚沈元曦纪荣毅周志平冒丽刘冀
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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