Avian leukosis double-antibody sandwich enzyme-linked immuno sorbent assay (ELISA) antigen detection kit

A double-antibody sandwich and antigen detection technology, which is applied in the field of biotechnology detection, can solve problems such as the impact on the production performance of chicken flocks, the economic loss of the poultry industry, and the decline in egg-laying performance, and achieves low cost, simple operation, and improved sensitivity. Effect

Inactive Publication Date: 2013-06-19
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

ALV has caused huge economic losses to the poultry industry due to the induction of tumors, the discarding of affected chicken carcasses, the decline in egg production performance and the impact on the performance of flocks
However, so fa

Method used

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  • Avian leukosis double-antibody sandwich enzyme-linked immuno sorbent assay (ELISA) antigen detection kit
  • Avian leukosis double-antibody sandwich enzyme-linked immuno sorbent assay (ELISA) antigen detection kit
  • Avian leukosis double-antibody sandwich enzyme-linked immuno sorbent assay (ELISA) antigen detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0028] 1. Preparation of monoclonal antibody

[0029] 1. Preparation of monoclonal antibody (see technical route figure 1 )

[0030] According to the published gene sequence of ALV JS-nt strain (accession number: HM235667), a pair of specific primers targeting p27 were designed and synthesized, and BamHI and XhoI restriction sites were added to the upstream and downstream respectively, provided by Shanghai Yingjun Company synthesis. The primer sequences are as follows:

[0031] Upstream primer 5'-GCGGATCCATGCCTGTAGTGATTAAGACAG-3' (SEQ ID NO.1);

[0032] Downstream primer 5'-GCCTCGAGTTAGGCCGCGGCTATGCCT-3' (SEQ ID NO. 2).

[0033]The p27 gene was amplified from the ALV genome by PCR, and the amplified fragment was cloned into the pGEX-6P-1 expression vector (product of GE Company), and the recombinant strain pGEX-6p-1-P27 / BL21 was screened. IPTG induces protein expression of GST-p27, and uses GE company protein prepacked column to purify the protein to obtain purified GST-p...

Embodiment 2

[0100] The composition of the kit in this example is as follows:

[0101] ELISA plate: coated with monoclonal antibody secreted by ALV P27-5D3 strain

[0102] Enzyme-labeled antibody: horseradish peroxidase-labeled monoclonal antibody 4F12 produced by ALV P27-4F12 secretion E

[0103] Substrate solution preparation: 100mmol / L citric acid solution (21g citric acid (C 6 h 8 o 7 H2O) was dissolved in deionized water, and the volume was adjusted to 1L) 24.3mL, 200mmol / LNa 2 HP0 4 .12H 2 0 (71.6g Na 2 HP0 4 .12H 2 0 dissolved in deionized water, dilute to 1L) 25.7mL and mix well, add 50mg of tetramethylbenzidine (TMB), add 50μL of 30%H 2 0 2 ;

[0104] Stop solution (2mol / LH 2 S0 4 ): Mix 177.8mL of distilled water and 22.2mL of concentrated sulfuric acid respectively.

[0105] Washing solution: add 0.5mL Tween-20 to 1000mL10mmo1 / L PBS with pH7.4;

[0106] Negative and positive controls.

[0107]

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Abstract

The invention provides an avian leukosis double-antibody sandwich enzyme-linked immuno sorbent assay (ELISA) antigen detection kit. The kit comprises ELISA plates which are coated on anti-avian leukosis virus (ALV) p27 monoclonal antibodies, enzyme labeling anti-ALVp27 monoclonal antibodies and the like. Antibody capturing and antibody detecting are respectively conducted aiming at different antigenic determinants of p27 protein; anti-ALVp27 monoclonal antibodies coated by the ELISA plates are obtained through secretion of hybridoma cell strain ALVP27-5D3, and the enzyme labeling anti-ALVp27 monoclonal antibodies are obtained through secretion of hybridoma cell strain ALVP27-4F12. The avian leukosis double-antibody sandwich ELISA antigen detection kit is easy, convenient and fast to operate, can be used in detecting of all subgroup virus of ALV, and is suitable for all levels of veterinarian departments in the basic level and rapid and mass screening detecting of the avian leukosis of leaving and entering the country. The avian leukosis double-antibody sandwich ELISA antigen detection kit has the advantages of being low in cost, notable in economical benefit, and wide in application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, in particular to a double-antibody sandwich ELISA antigen detection kit for detecting avian leukosis virus. Background technique [0002] Avian leukemia (AL) is a general term for a variety of neoplastic diseases caused by avian leukemia virus (ALV) in the retroviridae family, and it is distributed worldwide. According to the difference of virus host range, envelope characteristics and cross-neutralization reaction, ALV is divided into 10 subgroups A-J, of which E subgroup is endogenous ALV, and A, B, C, D and J subgroups are exogenous derived ALV. ALV has brought huge economic losses to the poultry industry due to the induction of tumors, the discarding of infected chicken carcasses, the decline of egg production performance and the impact on the performance of chicken flocks. However, so far, there are no preventive vaccines and effective drugs for the disease. Elimination of positive c...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 秦爱建钱琨尹丽平韶红霞金文杰
Owner YANGZHOU UNIV
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