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Sheep echinococcosis ELISA (enzyme-linked immunosorbent assay) antibody detection kit

A sheep echinococcosis and antibody detection technology, applied in the field of genetic engineering technology and diagnostic reagents, to achieve strong specificity, high sensitivity, and good stability of the kit

Inactive Publication Date: 2014-10-15
CHONGQING AULEON BIOLOGICALS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no simple, effective, specific, and sensitive immunological detection kit to evaluate the immune effect of vaccines and promote the application of EG95 recombinant protein vaccines

Method used

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  • Sheep echinococcosis ELISA (enzyme-linked immunosorbent assay) antibody detection kit
  • Sheep echinococcosis ELISA (enzyme-linked immunosorbent assay) antibody detection kit
  • Sheep echinococcosis ELISA (enzyme-linked immunosorbent assay) antibody detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Preparation of Sheep Echinococcus ELISA Antibody Detection Kit

[0023] Sheep Echinococcus ELISA Antibody Detection Kit includes Sheep Echinococcus Recombinant Antigen Eg95 Pre-coated ELISA Plate, Sample Diluent, Positive Control Solution, Negative Control Solution, Enzyme Conjugate, Washing Solution, Substrate Chromogenic Solution and Stop liquid.

[0024] The preparation method of sheep echinococcosis ELISA antibody detection kit is as follows:

[0025] 1. Expression and purification of echinococcus recombinant antigen Eg95: first PCR amplify the EG95 gene fragment, then use EcoR I and Xho I to simultaneously digest the PCR product and the prokaryotic expression vector pET-32a, and recover the target fragment and vector from the gel. Connected to construct the recombinant plasmid pET-EG95. The recombinant plasmids successfully identified by enzyme digestion and sequencing were transformed into E. coli BL21, positive colonies were screened and inserted i...

Embodiment 2

[0028] Example 2: Operation steps for the use of sheep Echinococcus ELISA antibody detection kit

[0029] 1. Take the antigen-coated plate, add 100 μl of the diluted serum to be tested and the control to the wells of the antigen-coated plate, make 1 well for the sample to be tested, set 2 holes for the negative control and positive control, 100 μl for each hole ; Gently shake the sample in the well (do not overflow), and incubate at 37°C for 60 minutes;

[0030] 2. Shake off the solution in the wells of the plate, add 200 μl of diluted washing solution to each well, let it stand for 3 minutes, pour it out, then pat dry on absorbent paper, and wash 5 times in total;

[0031] 3. Add 100 μl of rabbit anti-goat enzyme-labeled secondary antibody to each well, and incubate at 37°C for 60 minutes;

[0032] 4. Wash 5 times, the method is the same as 2, remember to pat dry on clean absorbent paper each time;

[0033] 5. Add 50 μl of substrate chromogenic solution A and 50 μl of sub...

Embodiment 3

[0037] Example 3: Echinococcus ELISA Antibody Detection Kit Test Result Evaluation

[0038] 1. Sensitivity test: Take a positive serum from 1:200 times and start doubling dilution, and then use this kit for ELISA test. When the serum dilution is 1:25600, the OD 450 =0.672, the result is still positive (see Figure 4 ), proving that the kit of the present invention has better sensitivity.

[0039] 2. Blocking test: use the coated antigen protein and known positive serum to carry out blocking reaction, and the positive serum

[0040] Divided into two parts, the first part was diluted 1:100 with the sample diluent, the second part was diluted 1:100 with the diluent containing 0.1μg / 100μl Eg95 antigen, and then the ELISA test was carried out according to the established operating procedures. The results are shown in Table 1. The results show that the positive serum treated with the coated antigen, OD 450 The value dropped significantly, indicating that the coated antigen has ...

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Abstract

The invention discloses a sheep echinococcosis ELISA (enzyme-linked immunosorbent assay) antibody detection kit. The detection kit comprises a sheep echinococcosis recombinant antigen Eg95 pre-coated ELISA plate, a sample diluent, a positive reference solution, a negative reference solution, an enzyme bonder, a scrubbing solution, a substrate color-developing solution and a stop solution. A preparation method of a sheep echinococcosis recombinant antigen Eg95 comprises the steps as follows: firstly, PCR (polymerase chain reaction) amplification is performed on an Eg95 gene segment, then EcoRI and XhoI are used for performing enzyme digestion on a PCR product and a prokaryotic expression vector pET-32a simultaneously, a target segment and the vector after gel recovery are connected to construct a recombinant plasmid pET-EG95, and the recombinant plasmid is transformed into Escherichia coli, induced by IPTG (isopropyl-beta-d-thiogalactoside) for expression and purified by Ni-NTA. According to the kit, recombinant protein Eg95 is used as the antigen to detect a sheep echinococcosis antibody, and the antibody detection kit is high in specificity, high in sensitivity and good in stability.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and diagnostic reagents, and in particular relates to a sheep echinococcosis ELISA antibody detection kit. Background technique [0002] Echinococcosis, also known as echinococcosis, is a serious zoonotic disease caused by the middle stage of Echinococcus granulosus parasitic in the small intestine of dogs, wolves, foxes and other animals-Echinococcus infects the intermediate host . Echinococcus parasitizes in the liver, lungs and other organs of domestic animals such as sheep, pigs, horses, camels, wild animals and humans. Due to the strong growth and large size of the larvae, it not only compresses the surrounding tissues to make them atrophy and dysfunction, but also easily causes secondary infection, such as the rupture of the larvae, and can also cause allergic reactions, often causing serious illness and even death to humans and animals. [0003] In 1996, Lightowler and others...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/535
CPCG01N33/6803G01N2333/43543
Inventor 曹政李阳春田东升李春燕
Owner CHONGQING AULEON BIOLOGICALS
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