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Double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting avian leukosis group specific antigen

A double-antibody sandwich and avian leukemia technology, which is applied in the field of detection of avian leukosis virus group-specific antigens, kits, and double-antibody sandwich ELISA kits, can solve the problem of no satisfactory diagnostic reagents for avian leukemia

Active Publication Date: 2012-08-15
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ELISA diagnostic kits for detecting the disease have been established in foreign countries for a long time, but there is no satisfactory commercialized diagnostic reagents for avian leukosis in China so far.

Method used

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  • Double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting avian leukosis group specific antigen
  • Double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting avian leukosis group specific antigen
  • Double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting avian leukosis group specific antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1 anti-fowl leukemia virus p27 protein monoclonal antibody and the preparation of the hybridoma cell line that secretes this antibody

[0057] 1. Materials and methods

[0058] 1.1 Viruses, experimental animals, cells and serum:

[0059] ALV strains ALV-J (HLJ09MDJ-1), ALV-A and ALV-B were isolated and identified by Harbin Veterinary Research Institute; IBV, MDV, AIV H5, AIV H7, AIV H9, ILTV, FPV, IBDV, NDV, ARV and REV were provided by Harbin Veterinary Research Institute; 3-week-old BALB / c mice, 2-month-old New Zealand rabbits and SPF chickens were provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; DF-1 and S / P20 cells were provided by the Preserved in the laboratory; rabbit anti-natural p27 protein positive serum was prepared by our laboratory.

[0060] 1.2 Carriers, bacteria:

[0061] Cloning vector pMD-18T, prokaryotic expression vector pET-30a, Escherichia coli DH5α and BL21 s...

Embodiment 2

[0097] The preparation of the polyclonal antibody of embodiment 2 anti-p27 protein

[0098] 1. Preparation of polyclonal antibodies

[0099] Choose 4 healthy female New Zealand white rabbits of about 2 kg, dilute the purified recombinant p27 protein (prepared according to the method described in Example 1) with PBS, add an equal volume of CFA to fully emulsify, and use 0.5 mg of recombinant protein per rabbit Inoculate subcutaneously at multiple points; after that, emulsify with the same amount of IFA every 2 weeks, boost immunization twice, collect 0.5ml of blood from the ear vein 7 days after the third immunization, and separate the serum.

[0100] 2. Detection of polyclonal immunogenicity by indirect immunofluorescence test

[0101] Spread DF-1 cells on a 24-well plate, add 100ul of ALV-A, ALV-B, and ALV-J viruses to each well, and set normal cells as negative controls. After continuing to culture in a 37°C incubator for 5 days, discard the supernatant and use Cells were ...

Embodiment 3

[0111] The establishment of the detection method of embodiment 3 avian leukosis virus group specific antigen

[0112] 1.1 Viruses and experimental animals

[0113] The virus strain HLJ09MDJ-1 (ALV-J) was preserved by our laboratory, 3-week-old BALB / c mice, 2-month-old New Zealand rabbits and SPF chickens were provided by the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0114] 1.2 Reagents and equipment

[0115] Protein A and Protein G columns were purchased from GE Company; immunofluorescence anti-rabbit secondary antibody, immunofluorescence anti-mouse secondary antibody, enzyme-labeled anti-rabbit secondary antibody were purchased from Sigma Company; TMB chromogenic solution was purchased from TianGen Company; enzyme-linked reaction plate was purchased from Jet Corporation.

[0116] 1.3 Purification of p27 monoclonal antibody and polyclonal antibody

[0117] Inject 1ml of 2E5-secreting hybridoma cells into...

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Abstract

The invention discloses double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting an avian leukosis group specific antigen. The kit comprises an enzyme plate coated by a monoclonal antibody which is secreted by a hybridoma cell strain the preservation serial number of which is CGMCC (china general microbiological culture collection center) No.5961. The invention also discloses a double-antibody sandwich ELISA method which is established by utilizing the monoclonal antibody and is capable of rapidly and effectively detecting an ALV (avian leukosis virus). In the double-antibody sandwich ELISA method, the monoclonal antibody prepared by pronucleus expressive HLJ09mdj-1p27 albumen is utilized as a peridium antibody, and antibodies prepared by p27 are utilized as a detection antibody. According to the invention, the minimum detection amount of the p27 is 1.25 ng / ml, the method is not reacted with the common virus of birds, and the specificity is good. The method is utilized to detect egg white and an anus swab sample, and the coincidence rate is respectively 96.5% and 88.9% compared with a PCR (polymerase chain reaction) method; and the result proves that the method has the advantages of convenience, celerity, differentia, sensitivity and the like, and is useful for the detection and population purification of the ALV.

Description

technical field [0001] The invention relates to a kit, in particular to a double-antibody sandwich ELISA kit for detecting the specific antigen of avian leukosis group and a method for detecting the specific antigen of the avian leukosis virus group by using the kit. Belongs to the field of biotechnology. Background technique [0002] Avian leukosis virus (ALV) belongs to the family Retroviridae and belongs to the genus of avian C-type retrovirus, which can cause various tumor diseases in poultry. The disease occurs all over the world, with a high infection rate and a low incidence rate, and is one of the main diseases that endanger the poultry industry. There are two main hazards of avian leukosis, one is to produce tumors, leading to the death of chickens; the other is to cause changes in production indicators such as hen egg production rate and egg weight reduction; moreover, it can also cause chicken immunosuppression, secondary Infection with other bacteria and viruse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577C12N5/20C07K16/10C12R1/91
Inventor 王笑梅高玉龙秦立廷祁小乐王永强高宏雷
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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