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Preparation method of ascitic-type avian influenza monoclonal antibody and hybridoma cell strain as well as avian influenza immunodetection kit

A hybridoma cell line and monoclonal antibody technology, applied in antiviral immunoglobulin, biochemical equipment and methods, measuring devices, etc., to achieve the effects of strong specificity of results, easy operation, and high accuracy

Inactive Publication Date: 2014-05-21
西安天星生物药业股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the establishment of avian influenza ELISA immunoassay kit with monoclonal antibody

Method used

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  • Preparation method of ascitic-type avian influenza monoclonal antibody and hybridoma cell strain as well as avian influenza immunodetection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The preparation of embodiment 1 ascites type avian influenza monoclonal antibody

[0046] 1. Immunogen and Sp20 mouse myeloma cell line

[0047] H9 subtype avian influenza vaccine (Veterinary Medicine (2011) 150132066) was selected as the immunogen. The Sp20 mouse myeloma cell line was purchased from the Cell Engineering Center of Fourth Military Medical University.

[0048] 2. Immunization of animals and immunity

[0049] Take 10 female 8-week-old Balb / C mice, and inject 0.2ml of bird flu vaccine into each mouse to immunize the mice intraperitoneally, and give additional immunizations on the 7th, 14th, and 28th days after the first immunization, respectively. On day 31, splenocytes were taken for cell fusion.

[0050] 3. Cell fusion and culture

[0051] (1) Preparation of immune spleen cells

[0052] ①Take a spleen from an immunized mouse, put it into a plate containing 5ml of incomplete culture medium (1640 culture medium without calf serum), put it on a 200-mesh...

Embodiment 2

[0086] Example 2 Avian Influenza Immune Detection Kit

[0087] 1. The composition of the avian influenza immunoassay kit

[0088] A 24-well culture plate

[0089] 22 sampling tubes

[0090] 25 droppers

[0091] 22 cotton swabs

[0092] Sample diluent (0.85M, pH9.6 carbonate buffer) 25ml

[0093] Positive control solution (sample diluent containing H9 subtype vaccine) 0.5ml~1ml

[0094] Negative control solution (sample dilution solution containing non-related proteins (such as normal bovine serum)) 0.5ml~1ml

[0095] Avian influenza monoclonal antibody liquid (PBS containing ascites-type avian influenza monoclonal antibody at a volume concentration of 1:3000 to 1:5000) 2.5ml~3ml

[0096] Enzyme-labeled antibody solution (PBS containing horseradish peroxidase-labeled goat anti-mouse IgG antibody at a volume concentration of 1:1000) 2.5ml~3ml

[0097] Chromogen A (phosphoric acid-citric acid buffer at pH 5.0 with a volume concentration of 0.045% hydrogen peroxide) 2.5ml~3...

Embodiment 3

[0115] 1. A chicken farm in Chang'an County, Shaanxi Province raised 3,000 laying hens. The breeding period was nearly two years. Recently, it was found that the chickens did not eat well, the egg production rate dropped, and occasionally some chickens died. They were tested for bird flu. The oral mucus of 10 chickens was randomly collected and tested with this immunoassay kit, and the results were all positive (Table 3). It shows that the whole chicken flock has bird flu virus, and some have been infected with bird flu disease.

[0116] Table 3 ELISA reaction result table

[0117] Sample (oral mucus)

Detection method

Test result (positive+, negative-)

1

ELISA

+

2

ELISA

+

3

ELISA

+

4

ELISA

+

5

ELISA

+

6

ELISA

+

7

ELISA

+

8

ELISA

+

9

ELISA

+

10

ELISA

+

[0118] 2. In the same chicken farm as in No. 1, aft...

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Abstract

The invention discloses a preparation method of an ascitic-type avian influenza monoclonal antibody and a hybridoma cell strain as well as an avian influenza immunodetection kit. According to the preparation method, the ascitic-type avian influenza monoclonal antibody is prepared. The hybridoma cell strain 1A7 which is capable of stably secreting the avian influenza (H9) subtype monoclonal antibody with high titer and strong specificity is successfully screened, and the collection number of the hybridoma cell strain is CGMCC No.8010. The avian influenza immunodetection kit is prepared based on the ascitic-type avian influenza monoclonal antibody secreted by the hybridoma cell strain. The kit has the main characteristics of strong specificity and high accuracy rate; by adopting the kit, only the mucus of mouths, noses and cloacae needs to be collected and serves as samples, and blood drawing is not needed; the kit does not cause damage to animals and stress reaction; the kit is simple and convenient to operate and can be used for simultaneously detecting batch samples, and reports can be reported in two and a half hours due to adoption of the kit; the kit can be used for early-stage detection and diagnosis of avian influenza on the scene without needing auxiliary equipment.

Description

technical field [0001] The invention relates to the field of veterinary biological products, in particular to a preparation method of ascites type bird flu monoclonal antibody, a hybridoma cell line and a bird flu immune detection kit. Background technique [0002] The full name of avian influenza is avian influenza, which is an animal infectious disease caused by a virus. Usually only birds and poultry are infected, but in rare cases it can also infect pigs and even humans. [0003] Avian influenza can be divided into three categories: non-pathogenic, low-pathogenic and highly pathogenic. Non-pathogenic avian influenza does not cause obvious symptoms, but only produces virus antibodies in the infected poultry and birds. Low-pathogenic avian influenza can cause mild respiratory symptoms, reduced food intake, decreased egg production, and sporadic deaths in poultry. Highly pathogenic avian influenza is very serious, with high morbidity and mortality. The mortality rate of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569
Inventor 黄威权赵锋朱秋融吕葆真李丹青
Owner 西安天星生物药业股份有限公司