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Monoclonal antibody of canine parainfluenza virus, and application thereof

A technology of canine parainfluenza virus and monoclonal antibody, applied in the biological field, can solve the problems of easy missed detection, economic loss, low sensitivity, etc., achieve good reactivity, overcome multiple strains of canine parainfluenza virus or easy to leak The effect of testing positive disease materials

Active Publication Date: 2019-11-05
LUOYANG PULIKE WANTAI BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there are few manufacturers of CPIV colloidal gold test strips. The main ones are the colloidal gold test card of Shanghai Kuailing Biotech Co., Ltd. and the canine parainfluenza virus colloidal gold test strip of Venture, but their sensitivity is low and easy to leak. detection, resulting in unnecessary economic losses, and none of these commercial kits can be detected on hosts other than dogs
[0004] At the same time, the current canine parainfluenza virus has undergone a certain degree of mutation (see literature Jae-Ku Oem et al. Molecular characteristics of canine parainfluenza viruses type 5 (CPIV-5) isolated in Korea. Canadian Journal of Veterinary Research, Volume 79, Number 1, January 2015, pp.64-67(4); and Ming Sun et al., Isolation and Identification of Canine Parainfluenza Virus and Partial Gene Sequence, Chinese Journal of Comparative Medicine, 2016.02, 26(2):77-82), among others Variations in antigens make it more difficult to effectively detect and accurately determine infection in animals

Method used

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  • Monoclonal antibody of canine parainfluenza virus, and application thereof
  • Monoclonal antibody of canine parainfluenza virus, and application thereof
  • Monoclonal antibody of canine parainfluenza virus, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Existing product testing situation of embodiment 1

[0047]60 clinically collected eye, nose and throat swabs of suspected canine parainfluenza virus-infected dogs were collected during the epidemiological investigation. RT-PCR, commercial canine parainfluenza virus colloidal gold test strip 1 (Kailing) and commercial test strip 2 (Venture) were used for detection respectively. The results (see Table 1) showed that RT-PCR was positive for 46 1 and 14 were negative, while commercial test strips 1 and 2 detected 21 and 24 positives respectively. The detection rates were high, indicating that the sensitivity of commercial test strips was low.

[0048] Table 1 Clinical sample detection results

[0049] Detection method or product Positive Negative RT-PCR 46 14 Commercial note 1 21 39 commercial note 2 24 36

Embodiment 2

[0050] The separation of embodiment 2 canine parainfluenza virus

[0051] Get RT-PCR detection positive among the embodiment 1, commercialization test strip detection is all negative 22 samples after filtering with 0.22 μm filter membrane, inoculate to about 80% Vero cells, add 10% fetus after absorbing 1 hour DMEM medium with bovine serum, 37°C, 5% CO 2 Continue to cultivate under the same conditions, and observe the cytopathic condition. After blind passage for 3 generations, the virus was harvested. The result: 18 samples had no obvious cytopathic changes after inoculation or the lesions were lost during the passage process, and only 4 samples could produce obvious cytopathic changes after inoculation. The four viral fluids harvested were designed with primers and amplified by molecular biology methods, followed by F gene sequencing. After comparison by NCBI, it was confirmed that they were all canine parainfluenza viruses, and they were named S0419, S0422, and S0427 respec...

Embodiment 3

[0053] Example 3 Preparation, Purification and Identification of Canine Parainfluenza Virus Monoclonal Antibody

[0054] 3.1 Immunization of mice

[0055] Four strains of canine parainfluenza virus isolated in Example 2 were used as immunogens, and emulsified in a conventional manner to immunize female BALB / c mice aged 4-6 weeks, 200 μl / mouse. From the second immunization, blood was collected 7 days after each immunization, and the antibody titer of mouse serum after immunization at different stages was detected by IFA method. The results (see Table 2) show that canine parainfluenza virus S0422 strain, S0443 strain 2 strains of virus liquid as immunogen immunized mouse serum does not produce titer, only S0419 strain, S0427 strain of virus liquid immunized mouse serum produces titer , and the IFA titer of the S0427 strain was the highest, so this group of immunized mice was selected for cell fusion to prepare monoclonal antibodies.

[0056] Table 2 Serum IFA titer of immunize...

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Abstract

The invention relates to murine monoclonal antibodies 2B7 and 4H1 of canine parainfluenza virus, and a variable region sequence of the monoclonal antibody can be specifically and conservatively bond with the canine parainfluenza virus. A colloidal gold detection kit prepared by using the murine monoclonal antibodies as an antibody pair can detect a variety of epidemic strains and standard strainsof the canine parainfluenza virus with high sensitivity, has a high coincidence rate with the RT-PCR detection method, and can meet the urgent clinical needs at present.

Description

technical field [0001] The invention relates to a canine parainfluenza virus monoclonal antibody, an antigen detection kit containing the monoclonal antibody and an application thereof, belonging to the field of biotechnology. Background technique [0002] Canine parainfluenza virus (CPIV) belongs to the Paramyxoviridae family and the genus Mumps virus. After infecting animals, cough and even fever are the main symptoms, which can cause pharyngitis, tonsillitis and bronchitis. The virus can infect dogs, foxes, minks, cattle, rabbits, monkeys, sheep, horses, poultry, guinea pigs, mice, hamsters, raccoon dogs, tigers and other animals (Liu Tengfei, Detection of canine parainfluenza virus and NP gene Studies on the cloning, expression and immunological activity of its recombinant protein, Xinjiang Agricultural University, Master's Dissertation, 2010), the positive rate of dogs, foxes, and minks is relatively high; among them, dogs are their natural hosts and can infect various ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577
CPCG01N33/56983G01N33/577
Inventor 田克恭郝丽影
Owner LUOYANG PULIKE WANTAI BIOTECH
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