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Double-antibody sandwich ELISA quantitative determination kit of porcine epidemic diarrhea virus and application

A porcine epidemic diarrhea and double-antibody sandwich technology is applied to antiviral immunoglobulins, specific peptides, and measuring devices. It can solve problems such as low sensitivity, inability to quantify, and complicated operations, and achieve good repeatability and easy operation.

Active Publication Date: 2017-08-29
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] From the winter of 2010 to the spring of 2011, severe infectious diarrheal diseases occurred successively in pigs in pig farms all over the country. The onset was sudden, the spread was fast, the epidemic range was wide, and the epidemic time was long. The application of various antibiotics was ineffective, and the incidence rate reached About 80%, the mortality rate is as high as 50% to 85%, causing major economic losses
[0004] Previously, conventional RT-PCR detection methods were mostly used for the detection of PEDV pathogens. The operation is complicated, time-consuming, costly, and cannot be quantitatively detected, only qualitatively judged, and cannot be used for high-throughput detection.
Another Korean colloidal gold test strip has low sensitivity, cannot be quantified, and is more expensive, so it is not suitable for high-throughput antigen detection

Method used

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  • Double-antibody sandwich ELISA quantitative determination kit of porcine epidemic diarrhea virus and application
  • Double-antibody sandwich ELISA quantitative determination kit of porcine epidemic diarrhea virus and application
  • Double-antibody sandwich ELISA quantitative determination kit of porcine epidemic diarrhea virus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, preparation recombinant N protein

[0029] 1. Sequence Synthesis

[0030] The published N gene sequence of PEDV was optimized according to the codon bias of Escherichia coli to obtain the optimized N gene sequence, as shown in SEQ ID NO:1. The amino acid sequence of N protein is shown in SEQ ID NO:2. The optimized N gene was sent to Gene Company for synthesis, and then the fragment was cloned into the prokaryotic expression vector pET-28a(+) between the BamH I and XhoI restriction sites to obtain the pET-28a-PEDV positive plasmid.

[0031] 2. Expression and purification of recombinant N protein

[0032] (1) Transformation: transform the prokaryotic expression host strain BL21(DE3) with the pET-28a-PEDV positive plasmid, spread it on LB solid medium containing kanamycin (kanamycin concentration 100 μg / mL), and culture at 37°C 12-16h. Pick a single colony and culture it in LB liquid medium containing kanamycin (kanamycin concentration 50μg / mL) for 12-16h...

Embodiment 2

[0037] Embodiment 2, preparation of mouse anti-PEDV N protein monoclonal antibody

[0038] 1. Immunogen preparation: The recombinant N protein (0.5 mg / mL) prepared in Example 1 was mixed with an equal volume of adjuvant to emulsify evenly, and it was in a water-in-oil state to prepare a recombinant N protein vaccine for immunizing mice. The adjuvants used for the first immunization were Freund's complete adjuvant and YOULONG adjuvant, and the adjuvants for the second and subsequent immunizations were Freund's incomplete adjuvant and YOULONG adjuvant.

[0039] 2. Immunization strategy: 4 Balb / c mice were subcutaneously immunized with recombinant N protein vaccine, and each mouse was subcutaneously immunized 3 times, each time each mouse was immunized with 0.1 mg of the immunogen, and the interval between each immunization was 4 weeks. One week after the third immunization, the blood was collected by tail docking, the serum was separated, and the antibody titer was detected by i...

Embodiment 3

[0065] Example 3, preparation of enzyme-labeled polyclonal antibody and antibody pairing screening

[0066] 1. Preparation of enzyme-labeled polyclonal antibody against porcine epidemic diarrhea virus N protein

[0067] (1) Preparation of polyclonal antibody against porcine epidemic diarrhea virus N protein

[0068] The recombinant N protein prepared in Example 1 was used to immunize rabbits according to conventional methods. When the antibody titer of rabbit serum (detected by the method in Example 2) reached above 1:243000, the rabbit serum was collected.

[0069] The polyclonal antibody was purified by the following method: the rabbit serum was centrifuged at 4000 rpm and room temperature for 15 min, and the supernatant was taken. Slowly add saturated ammonium sulfate solution drop by drop to the supernatant under stirring at 4°C until the saturation of ammonium sulfate in the system reaches 50%, continue stirring for 30 minutes, then centrifuge at 13,000 rpm and 4°C for 3...

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Abstract

The invention provides a double-antibody sandwich ELISA quantitative determination kit of a porcine epidemic diarrhea virus and an application and relates to the field of biodetection. A preservation number of a hybridoma cell strain PEDV of an anti-PEDV N protein monoclonal antibody is CCTCC NO: C201410. The double-antibody sandwich ELISA quantitative determination kit of the porcine epidemic diarrhea virus comprises a pre-coated elisa plate and an enzyme labeled antibody. The pre-coated elisa plate is the elisa plate coated by the anti-PEDV N protein monoclonal antibody, and the enzyme labeled antibody is a horse radish peroxidase labeled anti PEDV N protein polyclonal antibody; the anti PEDV N protein polyclonal antibody is obtained by immunizing a rabbit through the protein N of the porcine epidemic diarrhea virus to obtain serum of the rabbit. The kit provided by the invention is good in specificity, high in sensitivity and good in stability, can quickly, simply and efficiently detect the porcine epidemic diarrhea virus quantitatively, and is low in cost and suitable for high throughput detection.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a double-antibody sandwich ELISA quantitative detection kit for porcine epidemic diarrhea virus and its application. Background technique [0002] Porcine Epidemic Diahorrea Virus (PEDV) is the causative agent of porcine intestinal infectious disease, which can cause vomiting, diarrhea and dehydration in pigs of all ages. highest infection rate. The disease mostly occurs in the cold seasons of winter and spring, with December to February of the following year as the high incidence season. [0003] From the winter of 2010 to the spring of 2011, severe infectious diarrheal diseases occurred successively in pigs in pig farms all over the country. The onset was sudden, the spread was fast, the epidemic range was wide, and the epidemic time was long. The application of various antibiotics was ineffective, and the incidence rate reached About 80%, the mortality rate is as high as ...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/569C12R1/91
CPCC07K16/10C07K2317/35G01N33/56983G01N2333/165
Inventor 李彬何孔旺孙杰俞正玉范宝超郭容利茅爱华倪艳秀温立斌袁万哲张雪寒肖琦
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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