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Hybridoma cells capable of secreting substances resisting PEDV (porcine epidemic diarrhea virus) monoclonal antibodies, monoclonal antibody and application

A monoclonal antibody and hybridoma cell technology, applied in the biological field, can solve problems such as different antibody adaptability, strict quality requirements for colloidal gold, and weak fluorescent signals

Active Publication Date: 2019-08-20
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fluorescence method can achieve qualitative detection and its sensitivity is higher than that of colloidal gold, but aggregation is prone to occur in the fluorescently labeled antibody solution, and the fluorescent signal is weak and unstable
The surface-enhanced Raman immunoassay technology that has emerged recently has the advantages of high sensitivity and strong antibody specificity, such as the patent document "A Surface-Enhanced Raman Gold Standard Test Strip for Rapid Detection of Trace Fipronil and Its Preparation Method" (application No.: CN201810005898.X), but there are deficiencies in the strict requirements on the quality of colloidal gold and the different adaptability of Raman test strips to different antibodies

Method used

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  • Hybridoma cells capable of secreting substances resisting PEDV (porcine epidemic diarrhea virus) monoclonal antibodies, monoclonal antibody and application
  • Hybridoma cells capable of secreting substances resisting PEDV (porcine epidemic diarrhea virus) monoclonal antibodies, monoclonal antibody and application
  • Hybridoma cells capable of secreting substances resisting PEDV (porcine epidemic diarrhea virus) monoclonal antibodies, monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1 Obtaining of hybridoma cells and preparation of monoclonal antibodies thereof

[0079] 1. Preparation of PEDV virus

[0080] PEDV CHYJ130330 strain (GenBank: KJ020932, isolated, identified and preserved by Guangdong Haid Animal Husbandry and Veterinary Research Institute) was inoculated on a monolayer of Vero cells (purchased from ATCC, catalog number: ATCC CCL-81) at 37°C for 45 minutes. Then the maintenance medium (DMEM containing 1% (v / v) double antibody (penicillin mixture)) prepared by the laboratory was added to the culture for 2 or 3 days. When the cytopathy reached more than 85%, the cells were collected and repeated freezing and thawing 3 times. After sonication and centrifugation at 10000 rpm for 1 hour, the supernatant was further centrifuged at 45000 rpm for 3 hours, and the pellet was resuspended in PBS (0.01 M, pH 7.4). Crude virus extracts were layered in 30%-60% ((w / v)) sucrose-PBS in sequence, and centrifuged at 33000 rpm for 3 hours. Th...

Embodiment 2

[0113] Example 2 Application of Anti-PEDV Monoclonal Antibody—Double Antibody Sandwich Colloidal Gold Test Strip

[0114]1. Preparation of gold nanoparticles (AuNPs)

[0115] Scheme (1): AuNPs were prepared by referring to the Chinese book "Clinical Laboratory Experiment Series Tutorial----Immunological Laboratory Volume". Chlorauric acid (HAuClO 4 ) to make 0.005%-0.02% (w / v) aqueous solution first, take 100mL and heat to boiling. Immediately after boiling, 1 mL of 0.5% to 1.5% (w / v) trisodium citrate aqueous solution was accurately added under rapid stirring. Continue to heat and boil for 5 minutes, it turns orange red.

[0116] Scheme (2): optimize scheme (1), add 0.005%~0.02% (w / v) chloroauric acid and 0.5%~1.5% (w / v) trisodium citrate after boiling water, continue heating and boiling 5 ~ 15min, after stirring and cooling, dilute to 50mL with ultrapure water.

[0117] The AuNPs liquid surface prepared by scheme (1) has a lot of floating objects, poor light transmissio...

Embodiment 3

[0140] Example 3 Application of Anti-PEDV Monoclonal Antibody—Double Antibody Sandwich Fluorescent Test Strip

[0141] 1. Fluorescently labeled antibody

[0142] Scheme (1): Refer to the journal literature "Cui Hao, Chen Yaoqiang, Tang Yong, et al. Establishment of immunochromatographic detection method for ractopamine fluorescent latex particles [J]. Journal of Analysis and Testing, 2011,30(7):764-768 .” for fluorescently labeled antibodies.

[0143] Scheme (2): Optimizing Scheme (1), take 50 μL of fluorescent microspheres (purchased from Suzhou Weidu Biotechnology Co., Ltd., product number: FG02C) and dissolve them in 950 μL of 0.05M MES (2-(N-morpholine)ethanesulfonic acid) (pH 6.0), centrifuge at 15000rpm for 30min. The precipitate was blown away, dissolved in 1 mL of 0.05M MES (pH 6.0), and ultrasonically cleaned for 20 min. Add 10.65 μL 1mg / mL LEDC (carbodiimide), 6.65 μL 1mg / mL NHS (N-hydroxyperimide), mix well, rotate for 30 minutes, and ultrasonically clean for 20 ...

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Abstract

The invention provides hybridoma cells capable of secreting substances resisting PEDV (porcine epidemic diarrhea virus) monoclonal antibodies, a monoclonal antibody and an application. The hybridoma cells are respectively named as 4A11 and 3H7, are preserved in the China Center for Type Culture Collection, in Wuhan University, Wuhan, China on 13 June 2018, with the preservation numbers being CCTCCNO:C2018123 and CCTCC NO:C2018122. The hybridoma cell strains 4A11 and 3H7 can secrete monoclonal antibodies resisting porcine epidemic diarrhea viruses at a high yield, and ascites indirect-ELISA titer can reach 10<-6>. The porcine epidemic diarrhea virus detection test paper is also established by using the monoclonal antibody as a core, so that the porcine epidemic diarrhea viruses can be highly-specifically, accurately and sensitively detected, the operation is simple and convenient, effective and rapid site fast detection can be realized, and material support and technical support are provided for detection, diagnosis and scientific guidance on prevention and control of the porcine epidemic diarrhea viruses in China.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to hybridoma cells capable of secreting anti-PEDV monoclonal antibody, monoclonal antibody and application. Background technique [0002] Porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae and the genus Alphacoronavirus, is the causative agent of porcine epidemic diarrhea (PED), a highly contagious enteric disease whose main It is characterized by severe diarrhea in pigs, vomiting, dehydration and high morbidity in suckling piglets. Since 1982, PEDV has spread and spread in Southeast Asia, for example in Japan. In the past 30 years, the situation of PEDV infection in pig farms in Asia has been very serious. The disease has been further recorded in China and Thailand since 2005 and 2007 respectively. In the infected farms, pigs of all ages were affected. The effects of severe vomiting and watery diarrhea, and more seriously, the mortality rate of suckling pigl...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10C07K1/30G01N21/64G01N21/65G01N33/558G01N33/569
CPCC07K16/10C07K2317/35G01N21/6486G01N21/658G01N33/558G01N33/56983G01N2333/165
Inventor 唐勇边洪芬徐飞
Owner JINAN UNIVERSITY
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