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Double monoclonal antibody sandwich ELISA kit for detecting serotype 4 fowl adenovirus based on Fiber1 protein

A kit and a technology for detecting antibodies, applied in the field of biotechnology detection, can solve problems such as economic losses in the chicken industry, and achieve the effects of high sensitivity, good repeatability and strong specificity

Pending Publication Date: 2018-11-23
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is worth noting that in recent years, inclusion body hepatitis-pericardial effusion syndrome caused by serotype 4 avian adenovirus (FAdV-4) has become popular and broke out in domestic chicken flocks, causing serious economic losses to the chicken industry.
However, there is no effective vaccine against FAdV-4 and no commercialized antigen and antibody detection kits for rapid detection of serotype 4 avian adenovirus based on Spike protein 1.

Method used

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  • Double monoclonal antibody sandwich ELISA kit for detecting serotype 4 fowl adenovirus based on Fiber1 protein
  • Double monoclonal antibody sandwich ELISA kit for detecting serotype 4 fowl adenovirus based on Fiber1 protein
  • Double monoclonal antibody sandwich ELISA kit for detecting serotype 4 fowl adenovirus based on Fiber1 protein

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Experimental program
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Embodiment 1

[0023] Healthy Balb / c mice were taken, emulsified with purified Fiber1 protein and an equal amount of Freund's complete adjuvant, and then immunized, after the third immunization. Booster immunization was carried out within 72 hours of preparing for fusion to prepare for cell fusion. Before fusion, prepare 8 bottles of myeloma cells that are in the logarithmic phase of growth, and use DMEM without double antibodies and serum to blow down for later use.

[0024] Take the booster immunized mice, put them into 75% alcohol soaking for 10 minutes after the mice are killed by cervical dislocation, dissect the mice under aseptic conditions, find the spleen, carefully cut out the spleen, and put it into the pre-prepared containing double antibody In DMEM, remove the connective tissue around the spleen, put it into another plate containing DMEM, press the spleen with a curved needle, puncture the spleen with the needle, and completely release the spleen cells until the spleen cells tur...

Embodiment 2

[0026] (1) Preparation and Characterization of Monoclonal Antibodies

[0027] The capture antibody (anti-FAdV-4 spike protein 1 monoclonal antibody) coated on the microtiter plate was obtained from the secretion of the hybridoma cell line FAdV-4-F1-3B5, and the detection antibody was obtained from the hybridoma cell line FAdV-4-F1- 6H9 is secreted. Preparation of monoclonal antibodies to FAdV-4-F1-3B5 and FAdV-4-F1-6H9 ascitic fluid. Monoclonal antibody IgG was purified using Protein G prepacked columns from GE. SDS-PAGE was used to identify after purification, and the results showed that the correct and high-purity monoclonal antibodies were obtained. The SDS-PAGE identification of FAdV-4-F1-6H9 and FAdV-4-F1-3B5 monoclonal antibodies after purification figure 1 As shown, among them, lane M: prestained protein Marker; lane 1: purified FAdV-4-F1-3B5; lane 2: purified FAdV-4-F1-6H9; lane 3: unpurified FAdV-4 - F1-6H9 ascites; lane 4: unpurified FAdV-4-F1-3B5 ascites. Depend...

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Abstract

The invention relates to a hybridoma cell strain, an antibody and a kit for detecting FAdV-4 viruses based on a Fiber1 protein, and belongs to the technical field of biotechnology detection. The hybridoma cell strain for detecting the FAdV-4 viruses based on the Fiber1 protein comprises a hybridoma cell strain FAdV-4-F1-3B5 and a hybridoma cell strain FAdV-4-F1-6H9. A monoclonal antibody secretedby the hybridoma cell strain can realize double antibody sandwich efficient detection of the FAdV-4 viruses, is high in specificity and high in sensitivity, and is suitable for detecting trace antigens in a clinical specimen.

Description

technical field [0001] The invention relates to the technical field of biotechnology detection, in particular to a hybridoma cell line, an antibody and a kit for detecting FAdV-4 virus based on Fiber1 protein. Background technique [0002] According to genome characteristics and serum cross-test, avian adenovirus (Fowl Adenovirus, FAdV) can be divided into 5 species (A, B, C, D, E) and 12 serotypes (1-7, 8a, 8b, 9- 11). Epidemiological survey found that FAdV is susceptible to different poultry, and its acute infection mainly causes inclusion body hepatitis, pericardial effusion syndrome, and gizzard erosion. It is worth noting that in recent years, inclusion body hepatitis-pericardial effusion syndrome caused by serotype 4 avian adenovirus (FAdV-4) has become popular and broke out in domestic chicken flocks, causing serious economic losses to the chicken industry. However, there is no effective vaccine against FAdV-4 and no commercial antigen and antibody detection kits ba...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/569G01N33/577
CPCG01N33/543G01N33/56983G01N33/577
Inventor 叶建强鹿亚男邵红霞秦爱建王伟康董晓妹
Owner YANGZHOU UNIV