Preparation of egg antigen for detecting Ornithobilharziasis and ELISA detection kit

A technology for dongbi fluke and worm eggs is applied in the field of schistosome ELISA kits to achieve the effects of reducing the interference of human factors, simple operation, and simple and clear experimental results.

Pending Publication Date: 2019-12-03
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

It has not been reported that the egg antigen of oriental fluke is used as a diagnostic antigen

Method used

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  • Preparation of egg antigen for detecting Ornithobilharziasis and ELISA detection kit
  • Preparation of egg antigen for detecting Ornithobilharziasis and ELISA detection kit
  • Preparation of egg antigen for detecting Ornithobilharziasis and ELISA detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0027] The acquisition of the worm body and ovum of oriental fluke of embodiment 1

[0028] 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

[0031] Example 2 Extraction of worm egg soluble antigen:

[0032] 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

[0033] Example 3 Establishment of ELISA method for Orientobilax egg antigen

[0034] (1) Preparation of standard negative and positive serum

[0035]Ten samples of Tibetan goat serum that were positive for oriental birch disease in feces and necropsy were taken as standard positive serum; ten samples of normal goat serum from non-Oriental fluke endemic areas that were negative in fecal or necropsy were used as standard negative serum.

[0036] (2) Prepare egg soluble antigens as described in Example 2; use the same parameters to prepare adult worm soluble antigens.

[0037] (3) Determination of antigen coating concentration and serum dilution:

[0038] Soluble antigens of adult worms or eggs were diluted with antigen diluent to 0.5 μg / ml, 0.75 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 100 μl per well, coated overnight at 4°C, washed twice with PBST; Add 5% skimmed milk powder, 200 μl per well, incubate at 37°C for 1 h, wash with PBST 3 times; dilute the standard positive and negative ser...

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Abstract

The invention relates to a preparation of egg antigen for detecting Ornithobilharziasis and an ELISA detection kit. In the indirect ELISA detection method for Ornithobilharziasis established in the invention, the kit comprises Orientobilharzia egg antigen, a blocking solution (5% of skimmed milk powder), enzyme-labeled secondary antibody (HRP-labeled rabbit anti-goat Ig G), PBST, a color development solution (TMB substrate solution), and a stop solution (2mol/L of sulfuric acid), wherein the required antigen concentration is 0.5[mu]g/ml, and the serum dilution is 1: 100. The method of the application has the advantages such as high sensitivity, good accuracy and stability.

Description

technical field [0001] The invention relates to the technical field of protein purification and detection, in particular to a schistosomiasis ELISA kit prepared by using an oriental fluke egg antigen and an application thereof. 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. Oristania is subdivided into Ornsima pensdorf, A. schematica, A. turkestan and A. nodularis var. turkestan. Oriental flukes need to go through two hosts in the development process: the final host and the intermediate host. After burrowing into the skin of the final host cattle, sheep and other livestock, the cercariae of Orientobilax have migrated and finally developed into conjoined adults and laid eggs in the blood vessels of the host; The miracidia hatched in the medium; the miracidia enter the intermediate host snail snails for asexual reproduction, producing a large number of cerc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/543G01N33/58G01N33/53A01K67/033
CPCG01N33/535G01N33/543G01N33/5308G01N33/581A01K67/033
Inventor 朱传刚纪荣毅沈元曦周志平冒丽刘冀
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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