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Colloidal gold test strip for detecting sheep pox virus and preparation method thereof

A technology for colloidal gold test strips and sheep pox virus, which can be applied to measurement devices, instruments, scientific instruments, etc., and can solve the problems of complicated operation and time-consuming

Inactive Publication Date: 2016-08-17
王海艳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the establishment of the above method has played an important role in the prevention, diagnosis and monitoring of sheep pox, it is limited in on-site inspection and quarantine due to its complicated operation, time-consuming and special equipment. A method that is specific and can be used for on-site direct quarantine of sheeppox is imminent

Method used

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  • Colloidal gold test strip for detecting sheep pox virus and preparation method thereof
  • Colloidal gold test strip for detecting sheep pox virus and preparation method thereof
  • Colloidal gold test strip for detecting sheep pox virus and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Preparation of Preliminary Example 1 Rabbit Anti-Sheeppox Virus P32 Protein IgG

[0025] 1 Materials and methods

[0026] 1.1 Carriers and strains

[0027] The expression vector pET-21a plasmid and the expression strain Rosetta (DE3) were purchased from Shanghai Jiyin Biological Co., Ltd.

[0028] 1.2 Main reagents

[0029] Isopropyl-β-D-thiogalactoside (IPTG), Ex Taq enzyme, T4 DNA ligase, and BamHI and XholI restriction endonucleases were all purchased from Takara Company. Plasmid DNA extraction kit and gel recovery kit were purchased from Takara Company. His-tag Column affinity purification column was purchased from Sigma Company. LB medium was purchased from Beijing Land Bridge Company. Sephadex G200 chromatography column and PEG-20000 were purchased from Shanghai Chemical Reagent Factory. Complete Freund's adjuvant (CAF) and incomplete Freund's adjuvant (IAF) were purchased from GIBCO. Ammonium sulfate was purchased from Jinshan Chemical Factory, China.

[...

Embodiment 2

[0053] Preparation of Preliminary Example 2 Anti-P32 Protein Monoclonal Antibody of Goat Pox Virus

[0054] 1 Materials and methods

[0055] 1.1 Materials

[0056] 1.1.1 Antigens

[0057] See Preliminary Example 1 for the preparation and purification methods of goat pox virus P32 expressed protein.

[0058] 1.1.2 Cells and experimental animals

[0059] BALB / c mice, mouse monoclonal antibody subclass identification kit and SP2 / 0 myeloma cells were purchased from Shanghai Gil Biochemical Co., Ltd.

[0060] 1.1.3 Reagents and media

[0061] RPMI-1640, newborn calf serum (super), HAT salt (100×), HT salt (100×), goat anti-mouse IgG-HRP, penicillin, and streptomycin were purchased from GIBCOL; fusion agent 50% PEG, lymph Cell separation medium, colchicine, complete Freund's adjuvant and incomplete Freund's adjuvant were purchased from Sigma.

[0062] 1.1.4 Preparation of the main solution

[0063] 1640 basal medium: 1 bag of RPMI-1640 powder, add 2.0g sodium bicarbonate (NaH...

Embodiment 3

[0098]Preliminary Example 3 Preparation of Gold-labeled Monoclonal Antibody Against Sheeppox Virus P32 Protein

[0099] 1 Preparation of colloidal gold

[0100] Colloidal gold was prepared by trisodium citrate reduction method. The specific operation was as follows: measure 100mL of three-distilled deionized water with a volumetric flask, pour it into a flat-bottomed flask with a specification of 500.0mL, and place the flask on a magnetic heating stirrer for heating. Put a magnetic stirrer in the cover, turn on the stirring knob to an appropriate speed, turn on the heating knob, and heat to boiling. Add 1.0mL1%HAuCL 4 Solution, continue to heat for 2min, then quickly add 1% trisodium citrate solution at one time according to the volume shown in Table 1, and continue to heat. The light yellow chloroauric acid aqueous solution gradually turns from yellow to gray to black and finally to red or orange red within 2 minutes after the addition of trisodium citrate. After the solut...

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Abstract

The invention discloses a colloidal gold test strip for detecting goatpox virus and a preparation method thereof. The colloidal gold test strip for detecting goatpox virus comprises a sample pad, a colloidal-gold conjugate pad, a nitrocellulose membrane, an absorbent pad and a backing, wherein the sample pad, the colloidal-gold conjugate pad, the nitrocellulose membrane and the absorbent pad are successively adhered to a nonabsorbent support sheet; the colloidal-gold conjugate pad is a fiberglass membrane bonded to gold-labeled anti-goatpox virus P32 protein monoclonal antibody; and the nitrocellulose membrane has a detection line prepared by spraying of rabbit anti-P32 protein IgG and a quality control line prepared by spraying of goat anti-mouse polyclonal antibody. According to the invnetion, composition of a confining liquid used for preparation of the colloidal-gold conjugate pad and composition of a coating buffer solution used for preparation of the detection line and the quality control line are further optimized. It shows through specificity and sensitivity test results that the colloidal gold test strip for detecting goatpox virus has high specificity and high sensitivity.

Description

technical field [0001] The invention relates to a colloidal gold test strip, in particular to a colloidal gold test strip for detecting sheep pox virus and a preparation method thereof, belonging to the detection field of sheep pox virus. Background technique [0002] Goat pox is an acute, febrile, contagious disease of sheep or goats caused by sheep pox virus or goat pox virus (GTPV) of the genus Capipoxvirus. The main manifestations are fever, papules and herpes on the skin or mucous membranes of hairless or less hairy parts. Goat pox is the most serious of all animal pox diseases, with a high fatality rate and large economic losses, seriously affecting the development of international trade and sheep farming. In addition, this disease is also of great significance in public health. China, Mongolia, Sweden, and India all have reports of human infection with sheeppox virus, which has been highly valued by countries all over the world. Import and export of susceptible anim...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983G01N2333/065
Inventor 王海艳赵治国敖威华郭铁筝潘国卿高利李刚赵林立吴绍强周艳君
Owner 王海艳
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