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Acian Metapneumovirus antibody ELISA detection kit

A technology of avian metapneumovirus and detection kit, which is applied in the field of immunology, can solve the problems of insufficient sensitivity of antibody detection and hazards of transmission, and achieve the effects of promoting prevention research, good safety, and easy operation

Active Publication Date: 2013-10-23
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

The commercial aMPV ELISA detection kits on the market are from Europe and the United States, and are mainly used for the detection of serum antibodies. The antigen coated is the whole virus extracted after the virus is inoculated into cells, which often brings potential harm to the spread of the disease; In addition, these detection reagents are not sensitive enough for the detection of antibodies to specific subgroups such as subgroup A; especially, there is still no development of ELISA detection kits using expressed recombinant proteins as coating antigens
aMPV contains a variety of viral structural proteins, among which F protein is the main structural protein of the virus, which contains neutralizing epitopes that can protect the body against aMPV infection, but the F protein contains multiple transmembrane regions that affect the expression of the protein in the system recombinant expression of

Method used

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  • Acian Metapneumovirus antibody ELISA detection kit
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  • Acian Metapneumovirus antibody ELISA detection kit

Examples

Experimental program
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Embodiment 1A

[0049] Preparation and purification of the protein monomer of embodiment 1A subgroup avian metapneumovirus F protein

[0050] Artificially synthesize the gene sequence of the F protein of subgroup A avian metapneumovirus shown in SEQ ID NO.1, design a pair of specific primers with Oligo6.0 software, add KpnⅠ restriction site to the upstream primer, and add HindⅢ to the downstream primer Restriction site, the upstream primer is: 5'-GG GGTACC GAGGCAGTATCCACATTAGGG-3', the downstream primer is: 5'-CCC AAGCTT CTTGGCATCTGCACCTAG-3'. The pcmy-myc-L plasmid (purchased from Invitrogen) was used as a template to amplify in a 20 μL reaction system (ddH 2 (11.5 μL, 5×Buffer 4 μL, Taq enzyme 0.2 μL, dNTP 2 μL, upstream primer 1 μL, downstream primer 1 μL, DNA template 0.3 μL). The amplification conditions are: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 30 seconds, 59°C Anneal for 45s, extend at 72°C for 1.5min, a total of 35 cycles; extend at 72°C for 10min; store at ...

Embodiment 2

[0056] Example 2 Identification of the immunogenicity of the F protein monomer constructed by the present invention

[0057] Dilute the recovered F protein and undigested F protein with pH 9.6 0.5mol / L carbonate buffer solution to 10 μg / mL and coat a 96-well plate, and infect chicken positive serum and Negative sera were primary antibodies that were serially diluted in 3 gradients, 1:100, 1:300, and 1:500, respectively. Use HRP-labeled goat anti-chicken IgG as the secondary antibody, and the working concentration is 1:10000; do 2 wells in parallel for each sample, and read the OD of each well after color development by TMB 450nm The results show that: the protein without enterokinase digestion and carrier tag is used as the ELISA plate coating antigen, when the serum is detected, the OD value of the negative serum is between 0.6-0.8, which is close to the OD value of the positive serum; and After digestion with enterokinase, only the monomeric protein is retained as the coati...

Embodiment 3

[0060] The establishment of embodiment 3 avian metapneumovirus ELISA detection method

[0061] 1. Determination of the optimal reaction conditions of ELISA detection kit for subgroup A avian metapneumovirus antibody

[0062] 1) Determination of the optimal coating concentration of the antigen and the optimal working concentration of the enzyme-labeled monoclonal antibody

[0063] The purified F monomeric protein was used as the coating antigen, and the HRP-labeled goat anti-chicken monoclonal antibody was used as the second antibody, and the square array titration test was carried out on the enzyme plate. Dilute the F protein recovered by enzyme digestion with carbonate buffer to 15 μg / ml, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, respectively, and dilute 4 dilutions, each dilution is repeated for 2 wells, 100 μL / well, Coat the plate overnight at 4°C. The negative and positive sera of the chicken were diluted 1:100, 1:300, 1:500 respectively, and each dilution was repeated for 2 wells, ...

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Abstract

The invention provides an Acian Metapneumovirus antibody ELISA detection kit. The kit is the ELISA kit established for detecting the Acian Metapneumovirus antibody on the basis of treating the monomer protein of the A subgroup Acian Metapneumovirus F protein as a coating antigen. The invention also provides a preparation method of the monomer protein of the coating antigen F protein for making the kit. The F protein prepared through the method has a good reactionogenicity, and the established ELISA detection kit provides an effective method for the early-stage diagnosis and detection of the Acian Metapneumovirus infection, and plays a substantial promotion effect in the prevention researches of the Acian Metapneumovirus infected diseases.

Description

technical field [0001] The invention relates to the technical field of immunology, in particular to a monomeric protein of F protein of subgroup A avian metapneumovirus and an ELISA kit for detecting the antibody of subgroup A avian metapneumovirus. Background technique [0002] Avian Metapneumovirus (aMPV), also known as Turkey Rhinotracheitis Virus (TRTV), belongs to the Pneumovirus genus of the Paramyxoviridae Pneumoviridae subfamily, and is an important species of poultry distributed worldwide. pathogen. This virus mainly causes upper respiratory diseases of turkeys and chickens, such as turkey rhinotracheitis (TRT), avian rhinotracheitis (Avian Rhinotracheitis, ART), and is associated with broiler chicken swollen head syndrome (Swollen Head Syndrome, SHS) are closely related. Among them, turkey rhinotracheitis is an acute and highly contagious infectious disease that endangers the turkey farming industry. The main symptoms of the disease are runny nose, sneezing, swel...

Claims

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Application Information

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IPC IPC(8): C07K14/115C12N15/45C12N15/70G01N33/68G01N33/569
Inventor 刘爵阎旭韦莉王菁朱珊珊柳舒航张春燕全荣
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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