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Colloidal gold test strip capable of simultaneously detecting type-1 and type-3 duck hepatitis A viruses and preparation method of colloidal gold test strip

A duck hepatitis A, colloidal gold test paper technology, applied in the direction of measuring devices, instruments, biological material analysis, etc., to achieve the effect of good sensitivity and rapid detection technology

Inactive Publication Date: 2017-07-28
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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  • Colloidal gold test strip capable of simultaneously detecting type-1 and type-3 duck hepatitis A viruses and preparation method of colloidal gold test strip
  • Colloidal gold test strip capable of simultaneously detecting type-1 and type-3 duck hepatitis A viruses and preparation method of colloidal gold test strip
  • Colloidal gold test strip capable of simultaneously detecting type-1 and type-3 duck hepatitis A viruses and preparation method of colloidal gold test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the acquisition and identification of hybridoma cell line DHAV-MAb1

[0052] 1. Reproduction and purification of type 1 duck hepatitis A virus

[0053] 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.

[0054] Take the above-mentioned frozen duck embryo allantoic fluid, freeze and thaw repeatedly 3 times, centrifuge at 4°C, 6000r / min for 15min, discard th...

Embodiment 2

[0071] Example 2, Preparation and Identification of Monoclonal Antibody

[0072] 1. Mass preparation and purification of monoclonal antibodies

[0073] 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.

[0074] Take the purified ascites, and use SDS-PAGE to detect the purification effect of the monocl...

Embodiment 3

[0091] Embodiment 3, preparation and performance evaluation of colloidal gold test strip

[0092] 1. Preparation and purification of gold-labeled monoclonal antibodies

[0093] 1. Treatment of monoclonal antibodies

[0094] Put the ascites induced by the purified hybridoma cell line DHAV-MAb1 into a dialysis bag, dialyze overnight at 4°C in 0.01mol / L, pH7.4 PBS, change the dialysate every 4 hours, 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.

[0095] 2. Preparation of gold-labeled monoclonal antibody

[0096] 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 the monoclonal antibody that simultaneously recognizes duck hepatitis A virus type 1 and type 3, stirred and mix...

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Abstract

The invention discloses a colloidal gold test strip capable of simultaneously detecting type-1 and type-3 duck hepatitis A viruses and a preparation method of the colloidal gold test strip. The test strip consists of a bottom plate, a nitrocellulose membrane, a sample pad, a colloidal gold pad and an absorbing pad; anti-mouse IgG coats a quality control line on the nitrocellulose membrane; an anti-type-1 duck hepatitis A virus polyclonal antibody coats a detection line; and while colloidal gold coats the colloidal gold pad, the colloidal gold pad recognizes monoclonal antibodies of the type-1 and type-3 duck hepatitis A viruses. The test strip has high sensitivity, specificity, repeatability and stability, and therefore, a broad-spectrum, sensitive, specific, simple and rapid detecting technology which does not cause missing detection of the type-1 and type-3 duck hepatitis A viruses is provided for clinical and establishment units.

Description

technical field [0001] The invention relates to a colloidal gold immunochromatographic test strip and a preparation method thereof. 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. [0003] At present, there are many methods for detecting duck hepatitis A virus antigen, such as virus neutralization test, ELISA, RT-PCR, immunoelectron microscopy, etc., each ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/576G01N33/558G01N33/532
CPCG01N33/577G01N33/532G01N33/558G01N33/5768G01N2333/465
Inventor 程安春汪铭书杨旸
Owner SICHUAN AGRI UNIV
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