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An ibv-specific polypeptide antigen, antibody, ELISA detection kit and application thereof

A technology for detecting kits and polypeptide antigens, which is applied in the field of biotechnology detection, and can solve the problems of large differences between batches of antigens, the inability of the kits to detect antibodies of different serotypes, and the different cross-reaction capabilities.

Active Publication Date: 2021-04-06
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difference in antigens used, different test results often appear. Commonly used commercial ELISA kits use purified viruses and are properly treated for antigen coating. Some scholars use gene fusion expression proteins as coating antigens, but these The method often causes large batch-to-batch variability or non-specificity due to the purity of the antigen
The most important thing is that due to the different serotypes of IBV strains, the cross-reactive ability is also different. Sometimes the result of the entire test is changed due to the change of a few amino acids, so it often happens that the kit cannot detect antibodies of different serotypes.

Method used

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  • An ibv-specific polypeptide antigen, antibody, ELISA detection kit and application thereof
  • An ibv-specific polypeptide antigen, antibody, ELISA detection kit and application thereof
  • An ibv-specific polypeptide antigen, antibody, ELISA detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Using biological software to analyze the amino acid sequence of S2 of IBV, several possible antigenic fragments were determined from the hydrophilicity, antigenic index, surface site index, and conservation of S2 protein, respectively.

[0043] NO: 1, NCPYVSYGKFCIKPDGSIST

[0044]NO: 2, CGKKSSYYTTFDNDVVTEQYRPKKSVC

[0045] NO: 3, CSQQRELATQKINECVKSQSIRC

[0046] NO: 4, CNLTVTDEYIQTRMDKVQINCLQYICGNSLEC

[0047] NO: 5, CQQYGPVCDNILSVVNSVGQKEDMELLNFYSSTKPAGC

[0048] NO:6, SCPYVSYGRFCIQPDGSIKQ

[0049] NO:7, SCPYVSYGRFCIKPDGSIKQ

[0050] NO:8, SCPYVSYGRFCIQPDG

[0051] NO: 9, YVSYGRFCIQPDGSIKQ

[0052] Send NO: 1 to NO: 9 to Shanghai Jietai Biotechnology Co., Ltd. for synthesis of peptide antigens, and use the steps of IBV-specific antibody ELISA detection method to coat the above peptide antigens, specifically including the following steps:

[0053] 1) Coating the polypeptide antigens on the microtiter plates respectively, and blocking with rabbit serum after coati...

Embodiment 2

[0063] Due to the ELISA detection of OD value, it was found that the selected five-segment polypeptide, NO.1 polypeptide had the best reaction, and could react with M41, CK / CH / 2014 / FJ14, CK / CH / 2014 / QL1403, 4 / 91 strain serum, but with The serum of CK / CH / 2010 / JT1 strain did not react. Therefore, we selected the polypeptide sequence NO: 6SCPYVSYGRFCIQPDGSIKQ of CK / CH / 2010 / JT1 strain, and mutated one of the amino acids to obtain NO: 7SCPYVSYGRFCIKPDGSIKQ, and used the same method as in Example 1 to detect different genotype IBV sera. The results are the average value of OD650, the results are as follows:

[0064] Table 2

[0065]

Embodiment 3

[0068] According to the results of the ELISA detection OD value of Example 2, the CK / CH / 2010 / JT1 polypeptide antigen can react with the positive serum of each genotype IBV, and we shorten the most reactive polypeptide NO:6 to the following four amino acids as NO: 8SCPYVSYGRFCIQPDG, shortening the front three amino acids is NO:9YVSYGRFCIQPDGSIKQ, reacts with the positive serum of different strains IBV, all the results in the examples are the average value of OD650, the results are as follows:

[0069] table 3

[0070]

[0071]

[0072] The results of ELISAOD value showed that after peptide NO:6 shortened the amino acid, the OD650 value of peptide NO:8 and NO:9 had different degrees of reactivity with different genotypes of IBV positive sera, and did not react with 4 / 91 strains of positive sera. So the reactivity of peptide NO:8 and NO:9 is not as good as that of peptide NO:6.

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Abstract

The invention discloses a chicken infectious bronchitis virus (IBV) specific polypeptide antigen. The IBV specific polypeptide antigen has an amino acid sequence as shown in any one or several of SEQ ID NO: 1-9, or has An amino acid sequence formed by substitution, deletion or addition of one or more amino acid residues. The invention also discloses an IBV specific polypeptide antigen composition. The invention also discloses an IBV antibody and a preparation method thereof. The invention also discloses an ELISA detection kit, a detection method and application thereof. The antigen epitope has good antibody reactivity, can be used for specific detection of anti-IBV antibody, and has application value.

Description

[0001] Technical solutions [0002] The invention belongs to the technical field of biotechnology detection, and in particular relates to an IBV specific polypeptide antigen, an IBV antibody, an ELISA detection kit and applications thereof. Background technique [0003] Infectious bronchitis virus (IBV) is a γ-coronavirus, which can cause severe respiratory and kidney lesions in broiler chickens and drop in egg production in laying hens. At the same time, it spreads rapidly and brings huge economic losses to poultry production. Due to the special transcription mechanism of coronavirus, new mutant strains and serotypes are constantly produced, making the prevention and control of the disease very difficult. The methods currently used to detect IBV antibodies include IFA, ELISA, HI and other methods, and the latter two are suitable for large-scale detection. However, due to the difference in the antigens used, different test results often appear. The commonly used commercial EL...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/165C07K7/08C07K16/10G01N33/569
CPCC07K7/08C07K14/005C07K16/10C12N2770/20022G01N33/56983G01N2333/165
Inventor 秦爱建武奇钱琨邵红霞叶建强
Owner YANGZHOU UNIV