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Monoclonal antibody capable of simultaneously recognizing duck hepatitis A virus 1 and duck hepatitis A virus 3, and hybridoma cell strain and application thereof

A technology of hybridoma cell lines and monoclonal antibodies, applied in antiviral immunoglobulins, chemical instruments and methods, and methods based on microorganisms, to achieve the effects of strong specificity, good stability, and high affinity

Inactive Publication Date: 2017-06-09
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, both type 1 and type 3 duck hepatitis A have occurred and prevailed in duck production in my country, but to find common antibodies for type 1 and type 3 duck hepatitis A virus and establish a method that can simultaneously detect type 1 and type 3 duck hepatitis A The simple and rapid detection method of hepatitis virus has always been a problem that has not been well solved in academia and production

Method used

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  • Monoclonal antibody capable of simultaneously recognizing duck hepatitis A virus 1 and duck hepatitis A virus 3, and hybridoma cell strain and application thereof
  • Monoclonal antibody capable of simultaneously recognizing duck hepatitis A virus 1 and duck hepatitis A virus 3, and hybridoma cell strain and application thereof
  • Monoclonal antibody capable of simultaneously recognizing duck hepatitis A virus 1 and duck hepatitis A virus 3, and hybridoma cell strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, the acquisition and identification of hybridoma cell line DHAV-MAb1

[0039] 1. Reproduction and purification of type 1 duck hepatitis A virus

[0040]The virus solution of type 1 duck hepatitis A virus X strain (preservation number CCTCC NO: C201538) was properly diluted with normal saline, filtered through a 0.22 μm microporous membrane, and inoculated into 10-day-old duck embryos through the allantoic cavity , 0.1mL / piece; another 5 10-day-old duck embryos were taken as the control group and inoculated with normal saline; incubated at 37°C, discarded duck embryos that died within 24 hours of inoculation, and illuminated eggs twice a day for 3-5 days; The allantoic fluid of duck embryos that died 36-72 hours after inoculation and had obvious lesions was collected and frozen at -20°C for future use.

[0041] Take the above-mentioned frozen duck embryo allantoic fluid, freeze and thaw repeatedly 3 times, centrifuge at 4°C, 6000r / min for 15min, discard the...

Embodiment 2

[0058] Example 2, Preparation and Identification of Monoclonal Antibody

[0059] 1. Mass preparation and purification of monoclonal antibodies

[0060] BALB / c female mice aged 10-12 weeks were pre-sensitized with Freund's incomplete adjuvant, 0.5 mL / mouse, and 5 days later, 0.5 mL / mouse of hybridoma cell line DHAV-MAb1 (2.0×10 6 After 7-10 days, when the abdomen of the mouse was significantly enlarged, the ascites of the mouse was extracted, placed at 37°C for 2 hours, overnight at 4°C, centrifuged at 3000r / min for 15min the next day, and the supernatant was collected and stored at -20°C Save for later. The ascites can be extracted once every 2 days, and the mouse ascites can be extracted repeatedly for many times. Preserved ascites was first crudely extracted by octanoic acid-ammonium sulfate precipitation, and then purified by rProtein A / G Beads to obtain purified ascites.

[0061] Take the purified ascites, and use SDS-PAGE to detect the purification effect of the monocl...

Embodiment 3

[0078] Embodiment 3, preparation and performance evaluation of colloidal gold test strip

[0079] 1. Preparation and purification of gold-labeled monoclonal antibodies

[0080] 1. Treatment of monoclonal antibodies

[0081] Put the ascites induced by the purified hybridoma cell line DHAV-MAb1 into a dialysis bag, dialyze in 0.01mol / L, pH7.4 PBS overnight at 4°C, change the dialysate every 4h, and then centrifuge at 4°C and 10000r / min After 30 minutes, the supernatant was taken, and the protein content was determined to obtain the monoclonal antibody simultaneously recognizing type 1 and type 3 duck hepatitis A virus, which was aliquoted and stored at -20°C for later use.

[0082] 2. Preparation of gold-labeled monoclonal antibody

[0083] Take 1mL of 20nm colloidal gold solution, add 0.1mol / L K 2 CO 3 12 μL of the solution, stirred and mixed, added 34 μg of monoclonal antibody that simultaneously recognizes duck hepatitis A virus type 1 and type 3, stirred and mixed, stood...

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Abstract

The invention discloses a monoclonal antibody capable of simultaneously recognizing a duck hepatitis A virus 1 and a duck hepatitis A virus 3, and a hybridoma cell strain and application of the monoclonal antibody. The hybridoma cell strain is high in stability and can still secrete the monoclonal antibody after subculture in vitro and frozen storage and resuscitation of cells, the secreted monoclonal antibody can simultaneously recognize the duck hepatitis A virus 1 and the duck hepatitis A virus 3, the affinity constant with the duck hepatitis A virus 1 is more than 109 according to the order of magnitudes, the monoclonal antibody does not have specific reactions with the duck viral enteritis virus, the duck hepatitis B virus, the duck tembusu virus, the goose plague virus, the riemerella anatipestifer, the escherichia coli from ducks and the salmonella anatis, so that the broad-spectrum high-specificity high-affinity detection antibody is provided for clinics and basic units to avoid leak detection of the duck hepatitis A virus 1 and the duck hepatitis A virus 3, and can be used to prepare reagents for separately detecting the duck hepatitis A virus 1 or the duck hepatitis A virus 3 and simultaneously detecting the duck hepatitis A virus 1 and the duck hepatitis A virus 3.

Description

technical field [0001] The present invention relates to a monoclonal antibody, a hybridoma cell line secreting the monoclonal antibody, and the application of the monoclonal antibody. Background technique [0002] Duck hepatitis A virus (DHAV) can cause acute and highly fatal infectious diseases in ducklings, mainly infecting ducklings under 4 weeks of age, and the mortality rate of ducklings under 1 week of age is as high as 95%. Sick ducks have enlarged livers and hemorrhages. According to genome characteristics and antigen neutralization test, duck hepatitis A virus is divided into three genotypes or serotypes, namely duck hepatitis A virus type 1 (DHAV-1), type 2 (DHAV-2) and type 3 ( DHAV-3). Among them, duck hepatitis A virus type 1 and type 2 did not produce cross-protective effect, but had partial cross-neutralization reaction with type 3. At present, both type 1 and type 3 duck hepatitis A have occurred and prevailed in duck production in my country, but it is ne...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569C12R1/91
CPCC07K16/1009G01N33/56983G01N33/577G01N2469/10
Inventor 汪铭书程安春杨旸
Owner SICHUAN AGRI UNIV
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