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Immune colloidal gold test strip for rapidly detecting mucosal disease virus of sika deer

A technology for mucosal disease virus and colloidal gold test paper, which is applied to measurement devices, microorganisms, instruments, etc., can solve the problems of low specificity and cannot be applied on-site, and achieves the effects of strong specificity, convenient storage and low production cost.

Inactive Publication Date: 2012-10-31
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many methods to detect BVDV. In the early days, people used virus isolation, immunofluorescence technology, ELISA, etc. to detect BVDV. However, these methods have practical problems such as low specificity and inability to be applied in the field.

Method used

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  • Immune colloidal gold test strip for rapidly detecting mucosal disease virus of sika deer
  • Immune colloidal gold test strip for rapidly detecting mucosal disease virus of sika deer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1 BVDV polyclonal antibody purification

[0014] Take 10ml of New Zealand white rabbit serum immune to BVDV and mix it with an equivalent amount of 0.8% sodium chloride solution, then add 20ml of saturated ammonium sulfate solution dropwise to make a 50% saturated ammonium sulfate solution. Take it out at 4°C for 30 minutes, centrifuge at 3000rpm at 4°C for 30 minutes, discard the supernatant, add 20ml of 0.8% sodium chloride solution to the precipitate; after the precipitate dissolves, slowly add 10ml of saturated ammonium sulfate solution to make a 33% saturated ammonium sulfate solution . Take it out at 4°C for 30 minutes, centrifuge at 3000rpm at 4°C for 30 minutes, discard the supernatant, add 1ml of 0.8% sodium chloride solution to the precipitate, put it into a dialysis bag and dialyze with 0.8% sodium chloride solution to remove salt. Use 2% barium chloride solution to check whether the ammonium sulfate dialysis is complete. After complete dialysis,...

Embodiment 2

[0015] Example 2 Preparation of BVDV immune protein monoclonal antibody:

[0016] 1. Animal immunity

[0017] Firstly, the purified BVDV was completely emulsified with complete Freund's adjuvant, and then 5 healthy Balb / C mice aged 6-8 weeks were taken for a total of 3 immunizations. Intraperitoneally inject about 100 μg, and implement booster immunization 14 days later. The purified BVDV was emulsified with Freund's incomplete adjuvant for the second and third immunizations, and then each mouse was injected intraperitoneally, about 100 μg. 15 days after the third immunization, 100 μg of purified virus protein was injected intraperitoneally. About 3 to 4 days before the fusion, 50 μg of purified viral protein was injected into the tail vein.

[0018] 2. Establishment of hybridoma cell lines secreting anti-BVDV monoclonal antibody

[0019] The immune qualified mouse splenocytes were fused with SP2 / 0 myeloma cells, cultured by adding an appropriate amount of HAT-containing s...

Embodiment 3

[0022] Example 3 Preparation of gold-labeled monoclonal antibody

[0023] Colloidal gold solution was prepared by trisodium citrate reduction method. Add 1ml of chloroauric acid solution with a concentration of 10g / L to 99ml of three-distilled water and heat to boiling, then quickly add 2.4m / L 10.5g / L trisodium citrate solution, mix well, continue heating for about 5min-10min, the solution is Blue turns to red. Add an appropriate amount of purified anti-BVDV monoclonal antibody to the 50ml colloidal gold solution, stir at room temperature for 30min, slowly add 5ml 100g / L BSA and continue stirring for 30min. Centrifuge at 4000rpm for 10min, transfer the supernatant to another centrifuge tube, centrifuge for 30min, carefully discard the supernatant, and resuspend the pellet with 5ml 50mmol / LPBS. Soak 5×300mm glass fiber wool in the gold standard McAb solution, take it out and dry it in vacuum and store it in the dark.

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Abstract

The invention relates to an immune colloidal gold test strip for rapidly detecting the mucosal disease virus of sika deer. The immune colloidal gold test strip is characterized in that a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad of the test strip are sequentially adhered to a waterproof supporting slice; the combination pad is coated with a BVDV (Bovine Viral Diarrhea Virus) monoclonal antibody marked by colloidal gold; and the nitrocellulose membrane is respectively coated with a detection line T and a quality control line C, wherein the detection line T is formed by a BVDV purified polyclonal antibody, and the quality control line C is formed by stphylococcl protein A (SPA). The immune colloidal gold test strip has remarkable advantages of strong specificity, high sensitivity, simple operation and fast diagnosis and can be used for rapid detection of mucosal disease virus of sika deer and the diagnosis of epidemic diseases.

Description

technical field [0001] The invention relates to a colloidal gold test strip for rapidly detecting sika deer mucosal disease virus, which belongs to a new animal diagnostic reagent and is applied in the field of animal husbandry and veterinary medicine. The invention has far-reaching research and wide applicability, including epidemiological investigation of mucous membrane disease of sika deer in the region, purification of deer farms and rapid diagnosis of mucous membrane disease of sika deer. Background technique [0002] The causative virus of the mucosal disease of sika deer is bovine viral diarrhea virus (BVDV) / mucosal disease virus (MDV). The main features of the disease are nausea, vomiting, abdominal pain, diarrhea, draining or loose stools, leukopenia, mucosal ulcers, fever and general malaise, and more serious symptoms such as immune tolerance and immunosuppression, congenital defects, etc. In some cases, it can also lead to miscarriage of pregnant doe. There are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/558G01N33/531C12N5/18
Inventor 丁壮董航黄志强
Owner JILIN UNIV
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