Test for identifying and diangnosing enzyme-linked immunosobent of natural infective minnows for avian influenza

An enzyme-linked immunosorbent and natural infection technology, which is applied to measuring devices, instruments, scientific instruments, etc., achieves the effects of cumbersome operation, overcoming the inability to differentiate and diagnose avian influenza naturally infected flocks and vaccinated flocks, and low cost.

Inactive Publication Date: 2005-12-28
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the analysis of the prior art literature, the avian influenza NS1 protein was used as the coated antigen, and the level of antibody IgG reacting with the antigen in the chicken serum was detected by the method of indirect enzyme-linked immunosorbent assay, to differentially diagnose naturally infected chicken flocks and immunization Chicken flock, no related reports

Method used

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  • Test for identifying and diangnosing enzyme-linked immunosobent of natural infective minnows for avian influenza

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Prokaryotic Expression of H9N2 Avian Influenza Virus NS1 Protein

[0026] Cloning of H9N2 Avian Influenza NS1 Gene

[0027] Total RNA was extracted with Trizol (Invitrogen). Primers were designed according to the conserved sequence of avian influenza NS1 gene for RT-PCR amplification. Connected to the pMD-18T vector (Takara Company) for sequencing. According to the sequencing results, primers containing an EcoR I restriction site upstream and a Xho I restriction site downstream were designed for PCR amplification.

[0028] Construction of expression vector

[0029] EcoR I and Xho I respectively digested the PCR product and pET-32a (+), and T4 DNA ligase (Takara Company) was ligated overnight at 10-16°C. The ligated product was transformed into Escherichia coli DH5α, cultivated overnight at 37°C, and the plasmid was extracted. It was identified by EcoR I and Xho I digestion. Identified positive recombinant plasmids were sequenced.

[0030] Expression of fusion pro...

Embodiment 2

[0049] Establishment of Indirect ELISA Method

[0050] Determination of Optimal Enzyme-labeled Anti-IgG Antibody Concentration

[0051] The enzyme-labeled anti-IgG antibody was diluted from 1:5000 to 1:160000, and the optimal reaction concentration of the enzyme-labeled secondary antibody was measured to be 1:10000.

[0052] Optimal Antigen Reaction Concentration

[0053] Antigen according to the concentration of 100μg / ml, 80μg / ml, 60μg / ml, 40μg / ml, 30μg / ml, 15μg / ml, 10μg / ml, 8μg / ml, 6μg / ml, 4μg / ml, 2μg / ml, 1μg / ml For dilution, the antibody was diluted 1:50, 1:100, 1:500, 1:1000, and titrated in square array. The optimal antigen reaction concentration was determined to be 10 μg / ml.

[0054] Optimal Antibody Dilution

[0055] Dilute 20 negative sera, 11 vaccine-infected sera, and 12 natural infected sera 1:50, 1:100, 1:200, 1:500 times, and measure OD 450 , the test result is used as t test, when the difference between the negative serum and the vaccine infection serum is ...

Embodiment 3

[0059] Indirect enzyme-linked immunosorbent assay method for differential diagnosis of avian influenza virus in naturally infected chicken flocks.

[0060] After the antigen used was diluted to 10 μg / ml with coating diluent, 100 μl was added to each well, overnight at 4°C, and 2h at 37°C. Discard the liquid in the well.

[0061] Select 5% skimmed milk to block at 37°C for 2 hours, discard the liquid in the well, and wash with PBST washing solution 7 times.

[0062] Serum samples to be tested were diluted 1:200 times, 100 μl was added to each well, 37° C. for 1 hour, the liquid in the wells was discarded, and washed 10 times with PBST.

[0063] Add 100 μl of enzyme-labeled anti-IgG antibody, 37 ° C for 1 h, discard the liquid in the well, and wash 10 times with PBST washing solution.

[0064] Add 100 μl of TMD to develop color at room temperature for 8 minutes.

[0065] Add 50 μl of stop solution to stop the reaction.

[0066] Determination of OD 450 . judgement result.

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Abstract

A method of utilizing enzyme - linked immunosorbent assay to diagnose and identify naturally chicken herd of avian flu uses pronucleus to present NS 1 protein of H9N2 avian flu virus and uses it as antigen to coat reaction plate of enzyme mark. The method can identify and diagnose naturally infected chicken herd of avian flu and immunization chicken herd by using indirect enzyme - lined immunosorbent assay to determine level of antibody IgG in serum to be tested in reaction with antigen.

Description

technical field [0001] The invention relates to a method in the field of biotechnology, in particular to an enzyme-linked immunosorbent assay method for differential diagnosis of avian influenza naturally infected chicken flocks. Background technique [0002] The epidemic of highly pathogenic avian influenza has caused a large number of poultry deaths and poultry product losses. At present, one of the methods to effectively control bird flu is vaccination, and an index to effectively evaluate the immune effect is to detect the antibody level after immunization. However, in the late stage of wild virus infection, antibodies will also be induced, and at this time the chickens are already infected, and decisive measures must be taken in production. Therefore, it is very important to effectively distinguish antibodies produced by natural infection from those produced by immunization. In the early stage of avian influenza virus-infected cells, a large amount of NS1 protein can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535
Inventor 孙建和王琰陆承平严亚贤陆苹
Owner SHANGHAI JIAO TONG UNIV
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