Colloidal gold strip for TGEV antibody and PEDV antibody

A colloidal gold and antibody technology, which is applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of low specificity and low sensitivity, and achieve the effect of strong specificity, ensuring specificity and sensitivity, and avoiding virus operations.

Active Publication Date: 2013-04-10
兆丰华生物科技(南京)有限公司 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of whole virus and SPA has the characteristics of low sensitivity and low specificity. If the strong antigenic protein in the virus and its monoclonal antibody are used for a long time, the problem of sensitivity and specificity can be solved.

Method used

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  • Colloidal gold strip for TGEV antibody and PEDV antibody
  • Colloidal gold strip for TGEV antibody and PEDV antibody
  • Colloidal gold strip for TGEV antibody and PEDV antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 PEDV S 1 Expression and purification of protein and preparation of monoclonal antibody

[0031] 1. PEDV S 1 Protein expression and purification

[0032] ① Extraction and amplification of the target gene

[0033] The gold standard protein selected by this colloidal gold test strip is the fragment S with better reactogenicity in the PEDV S protein. 1 Protein, for domestic popular strains, according to its corresponding S 1 The following primers were designed for the protein gene

[0034] Upstream primer: 5'-ACGGATCCGATGGCACCAGGTGATC-3'

[0035] Downstream primer: 5'-CTGAATTCATGACTGTCCATGACTC-3'

[0036] The RNA of porcine epidemic diarrhea virus was extracted using a kit (ROCHE kit). Specific operation steps: Add 200 μL of virus liquid and 400 μL of Binding Buffer containing Poly A to a 1.5 mL EP tube, mix well and let stand for 1 min, then transfer to a high-efficiency filter tube, centrifuge at 8000×g for 15 s to discard the liquid; add to the filter tu...

Embodiment 2T

[0067] Example 2 Expression and purification of TGEV S recombinant protein fragment and preparation of its monoclonal antibody

[0068] 1. Expression and purification of recombinant expressed protein at AD site of S protein

[0069] ① Extraction and amplification of the target gene

[0070] The A and D sites with better reactogenicity in the TGEV S protein were selected for expression, and the following primers were designed for the recombinant expression protein gene of the AD site of the S protein according to GenBank comparison:

[0071] Recombinant expression protein of TGEV S protein AD site:

[0072] Upstream primer: ACGTCGACTGCTACGATGATTTCA

[0073] Downstream primer: GTAAGCTTATAGCCCCAACTATGG

[0074] Extraction of RNA is a routine operation of ROCHE kits. Use the kit (ROCHE kit) to extract the RNA of porcine transmissible gastroenteritis virus: add 200 μL of virus liquid and 400 μL of Binding Buffer containing Poly A to a 1.5mL EP tube, mix well and let stand for 1...

Embodiment 3

[0106] The assembly of embodiment 3 colloidal gold test strips

[0107] 1. Colloidal gold labeled antigen protein

[0108] Prepare gold solution by sodium citrate reduction method: add 4ml of 1.2% trisodium citrate solution to 50ml of boiling 0.02% aqueous auric acid chloride solution, continue stirring and heating until the solution is stable and bright red. Observing the colloidal gold solution under a transmission electron microscope, it is found that the colloidal gold particles are uniform in size, without oval or other irregular shapes, indicating that stable colloidal gold has been obtained.

[0109] With 0.1mol / L K 2 CO 3 Adjust the pH value of the colloidal gold to 7.5, and the protein to be labeled (S 1 or S T ) into the colloidal gold solution with adjusted pH value, and after standing for 10 minutes, add 20% polyethylene glycol (PEG) 10000 to a final concentration of 0.05%, and centrifuge at 2000r / min for 20 minutes at 4°C to remove unbound colloids Gold parti...

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Abstract

The invention discloses a test strip for quickly detecting a PEDV (Porcine Epidemic Diarrhea Virus) serum antibody and a TGEV (Transmissible Gastroenteritis Virus) serum antibody and a preparation method of the test strip. The test strip comprises a support layer, a sample loading layer, a gold labeling protein release pad, a detection layer and an absorption layer, wherein a gold labeling protein and a detection line protein of the test strip are an S1 protein of a PEDV and a recombination protein of a TGEV, which are obtained by an efficient prokaryotic expression system; the recombination protein comprises an S protein AD site; and two quality control line proteins are monoclonal antibodies for the two proteins. Compared with the traditional test strip for detecting the PEDV antibody and the TGEV antibody, the test strip is high in specificity and safety and simple to operate, and judges results quickly.

Description

technical field [0001] The invention belongs to the field of veterinary biotechnology, and relates to a rapid detection antibody technology, in particular to a colloidal gold detection test strip for two antibodies of porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). The invention relates to a preparation method and an application method of the test strip. Background technique [0002] Porcine transmissible gastroenteritis (Transmissible Gastroenteritis, TGE) and porcine epidemic diarrhea (Porcine Transmissible Gastroenteritis, PED) are caused by porcine transmissible gastroenteritis virus (Transmissible Gastroenteritis virus TGEV) and porcine epidemic Porcine Transmissible Gastroenteritis virus (Porcine Transmissible Gastroenteritis virus, PEDV) is a highly contagious enteric disease in pigs. It is mainly characterized by the beginning of autumn, the intensification of winter and spring, and the lack of summer. Pigs of all ages c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
Inventor 于倩倩王贵华于萍萍侯艳红陈翠云赵亚荣
Owner 兆丰华生物科技(南京)有限公司
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