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ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting porcine epidemic diarrhea virus antibody

A porcine epidemic diarrhea and kit technology, applied in the direction of virus/bacteriophage, virus, virus peptide, etc., can solve the problems of high morbidity and mortality, long epidemic time, economic loss, etc., and achieve good repeatability, easy operation, Detect accurate effects

Inactive Publication Date: 2014-08-06
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the winter of 2010 to the spring of 2011, serious 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. High mortality rate and significant economic losses
The ELISA method has the advantages of simplicity, rapidity, and strong specificity, and the ELISA detection antibody can evaluate the immune effect of the vaccine, but it requires high purity of the antigen and good immunogenicity

Method used

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  • ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting porcine epidemic diarrhea virus antibody
  • ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting porcine epidemic diarrhea virus antibody
  • ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting porcine epidemic diarrhea virus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1: specificity experiment

[0085] According to the established indirect ELISA method, the known PEDV, CSFV, PRRSV, PRV, TGEV, FMDV, PCV2 positive sera (provided by IDEXX kits) were detected respectively, and each serum was replicated three times in parallel. The OD value of each well was measured with a microplate reader at a wavelength of 450 nm to determine the specificity of the indirect ELISA method.

[0086] The results showed that the average S / P value of only PEDV-positive serum was 1.267>0.26, and the S / P values ​​of other serums were all between 0.051-0.187, far less than 0.26. It shows that the specificity of the detection results of the indirect ELISA method is good.

Embodiment 2

[0087] Embodiment 2: repeatability experiment

[0088] 1. Repeat experiment within batch:

[0089] Use the PEDV N protein prepared by induction and purification in the same batch, coat the microtiter plate according to the optimal antigen coating concentration, and use the established ELISA operation method to detect 8 randomly selected sera and standards with different levels of PED antibodies at different times For serum, the OD value was measured with a microplate reader at a wavelength of 450 nm. Calculate the S / P value of each serum, and the mean X, standard deviation SD, and coefficient of variation CV of the S / P value.

[0090] The results showed that the coefficient of variation of the S / P values ​​of the 8 serum samples was between 1.00% and 8.00%, and the results of the same batch of protein antigens operated at different times had good repeatability.

[0091] Table 3 Experimental results of intra-batch repeatability

[0092]

[0093] 2. Repeat experiment bet...

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Abstract

The invention provides an ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting a porcine epidemic diarrhea virus antibody, belonging to the technical field of biology. The kit is an ELISA plate stripe using purified recombinant porcine epidemic diarrhea virus N protein as an envelope antigen. RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification is adopted for obtaining a porcine epidemic diarrhea virus N gene, the gene is directionally inserted into a pET-28a carrier to construct pET-28a-N recombinant plasmids, BL21 (DE3) competent cells is transformed, and finally a prokaryotic expression fungus pET-28a-N / BL21 is obtained. The prokaryotic expression fungus pET-28a-N / BL21 is subjected to induced expression and purification through IPTG (Isopropyl Thiogalactoside) to obtain recombinant N protein. The kit is used for detecting the porcine epidemic diarrhea virus antibody. By testing, the kit has good specificity, sensibility and repeatability. The kit is accurate in detection, easy and convenient to operate and suitable for promotion in clinical application and provides a reliable means for rapid detection of porcine epidemic diarrhea.

Description

technical field [0001] The invention relates to an ELISA kit for porcine epidemic diarrhea virus antibody and application thereof, belonging to the field of biotechnology. Background technique [0002] Porcine Epidemic Diarrhea (PED) is an acute, highly contagious infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV), often characterized by acute enteritis and dehydrated water samples Diarrhea is a highly contagious intestinal infectious disease characterized by diarrhea, susceptibility to pigs of all ages and high lethality to suckling piglets. From the winter of 2010 to the spring of 2011, serious 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. Mortality is high and significant economic losses are caused. Studies have shown that porcine epidemic diarrh...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
CPCC07K14/005C12N2770/24322G01N33/56983G01N33/68G01N2333/183
Inventor 李彬孙冰何孔旺杜露平郭容利倪艳秀温立斌俞正玉张雪寒茅爱华吕立新胡屹屹周俊明王小敏祝昊丹于洋
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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