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ELISA kit for detection of highly pathogenic PRRSV infection

A highly pathogenic, kit-based technology, applied in the biological field, can solve problems such as high price and achieve high specificity and high sensitivity.

Active Publication Date: 2022-07-01
广东永顺生物制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of PRRSV antibodies in my country mainly relies on imported detection kits, which are expensive. Therefore, the establishment of domestic PRRSV antibody detection methods with good specificity and high sensitivity is of great significance for the prevention and control of PRRSV in my country. The cloned antibody is of great significance to the detection method and the preparation of the detection kit of PRRSV

Method used

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  • ELISA kit for detection of highly pathogenic PRRSV infection
  • ELISA kit for detection of highly pathogenic PRRSV infection
  • ELISA kit for detection of highly pathogenic PRRSV infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Establishment, identification and preservation of monoclonal antibody hybridoma cell lines

[0025] 1.1 Antigen preparation and immunization of Balb / c mice

[0026] The present invention adopts PRRSV virus NVDC-JXA1 strain (preserved and provided by Guangdong Yongshun Biopharmaceutical Co., Ltd.) as an immunogen, and inoculates PRRSV virus NVDC-JXA1 strain into Marc-145 cells (provided by Guangdong Yongshun Biopharmaceutical Co., Ltd.) In preservation and provision), when the cytopathic effect reaches 80-90%, freeze and thaw at -80°C for 3 times to release the virus from the cells, centrifuge at 3000 rpm for 5 minutes, remove cell debris, collect the virus liquid, and measure TCID50 Toxic price, aliquot and store at -70°C for later use.

[0027] The PRRSV virus NVDC-JXA1 strain frozen at -70°C was used as the immunization antigen, and it was diluted to 10 with sterilized normal saline. 6 TCID 50 / mL, and then mixed and emulsified with an equal volume of Freu...

Embodiment 2

[0061] Example 2: Preparation and purification of ascites fluid of hybridoma cell line 3E8 monoclonal antibody

[0062] 2.1 Preparation of monoclonal antibodies by inducing ascites in vivo

[0063] Selected female Balb / c mice, intraperitoneally injected with sterilized paraffin 500μL / mice, and one week later, intraperitoneally injected the obtained monoclonal hybridoma cell 3E8 again, with an injection volume of 10 6 After another week, the ascites was extracted after the abdomen of the mice was enlarged, and the supernatant was collected after centrifugation, and the ascites was purified by the saturated ammonium sulfate method.

[0064] 2.2 Purification of monoclonal antibody ascites (saturated ammonium sulfate method) and determination of titer

[0065] (1) Take the monoclonal antibody crude ascites, add an equal volume of PBS with pH=7.4, and mix the two gently.

[0066] (2) Place on a shaker, rotate gently at room temperature, slowly and dropwise add an equal volume of ...

Embodiment 3

[0072] Example 3: Activity identification of monoclonal antibodies

[0073] (1) Identification of strain detection activity of monoclonal antibodies

[0074] The PRRSV strain PRRSV-NVDC-JXA1 strain (virulent strain), PRRSV-GD strain (virulent strain), PRRSV-JXA1-R vaccine attenuated strain (attenuated strain of our company) kept by Guangdong Yongshun Biopharmaceutical Co., Ltd. Vaccine production strain), PRRSV virus classic strain PRRSV-VR2332 and European PRRSV classic strain PRRSV-DV strain infected Marc-145 cells, with uninfected Marc-145 cells, mouse negative serum, SP2 / 0 cells on Clear is the negative control, and the positive serum of the immunized mice is used as the positive control to analyze the reaction between the monoclonal antibody secreted by the purified hybridoma cell line 3E8 and the PRRSV strains. The specific results are as follows: figure 2 shown.

[0075] based on figure 2 The displayed results show that the monoclonal antibody secreted by the hybri...

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Abstract

An ELISA kit for detecting highly pathogenic PRRSV infection, said kit comprising: a microtiter plate coated with an anti-PRRSV polyclonal antibody, an anti-PRRSV monoclonal antibody, HRP-labeled goat anti-mouse IgG, a washing solution, Chromogenic solution, stop solution, sample lysate, positive control and negative control, characterized in that: the anti-PRRSV monoclonal antibody is secreted by hybridoma cell line 3E8. The kit can detect the currently popular American-type PRRSV virus, is suitable for the prevention and control of domestic PRRS epidemics, and can also detect the European-type PRRSV virus that is popular in Europe, and is suitable as the main detection method for import and export quarantine departments. , the lowest detected concentration of purified PRRSV-NVDC-JXA1 was 0.05 μg / mL, and the concentration of PRRSV-DV strain was 0.1 μg / mL, and the consistency with RT-PCR was 100%.

Description

Technical field: [0001] The invention belongs to the field of biology, and in particular relates to an ELISA kit for detecting highly pathogenic PRRSV infection and application thereof. Background technique: [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is a highly contagious disease caused by PRRS virus (PRRSV), characterized by reproductive disorders in pregnant sows and respiratory diseases in piglets. The main clinical symptoms are Reproductive disorders in pregnant sows, manifested as abortion, stillbirth, mummified fetuses, weak fetuses, respiratory diseases in pigs of all ages, especially in piglets, high mortality of suckling piglets, immunosuppression and persistent infection, etc., are currently seriously affecting the world's animal husbandry One of the important infectious diseases for the healthy development of the pig industry, especially since 2006, the highly pathogenic porcine reproductive and re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/58G01N33/569G01N33/543C07K16/10C07K16/06C07K1/22
CPCG01N33/577G01N33/581G01N33/56983G01N33/54306C07K16/10C07K16/065G01N2333/08
Inventor 陈坚林德锐涂玉蓉齐冬梅郑铁锁侯高伟陈根植尚书文钟植文黄艳珍
Owner 广东永顺生物制药股份有限公司