Dot immunogold filter kit for detecting IBR (infectious bovine rhinotracheitis) virus antibody and detection method thereof

A gold standard immune diafiltration and kit technology, applied in the field of gold standard immune diafiltration kits, can solve the problems of cumbersome inspection process, high detection cost, high price, etc., and achieve easy operation, convenient results, and intuitive effects Effect

Active Publication Date: 2012-09-26
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inspection process is cumbersome, virus detection takes 5d-6d to report a positive result, and the fastest ELISA method among serum antibody detection methods also takes 2h-4h
Moreover, most of the current reagents for detecting IBR antibodies are ELISA diagnostic kits imported from abroad, which are expensive and push up the detection cost. At the same time, specific instruments such as microplate readers, experimental skills of the operator, and certain experimental environmental conditions are required. Promotion in grassroots clinics

Method used

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  • Dot immunogold filter kit for detecting IBR (infectious bovine rhinotracheitis) virus antibody and detection method thereof
  • Dot immunogold filter kit for detecting IBR (infectious bovine rhinotracheitis) virus antibody and detection method thereof

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Effect test

preparation example Construction

[0028] 2. Preparation of goat anti-bovine antibody colloidal gold

[0029] 1) Preparation of nano-colloidal gold: Nano-colloidal gold sol was prepared by citric acid reduction method. Add 1 mL of 10mL / L chloroauric acid (purchased from Shanghai Reagent General Factory Chemical Reagent No. Heat the flask of the device to boiling, quickly add 2 mL of 10g / L trisodium citrate (purchased from Sinopharm Chemical Reagent Co., Ltd.) aqueous solution under magnetic heating and stirring, and continue heating and boiling until the solution turns wine color. The diameter of the obtained colloidal gold particles is (20-30) nm. After cooling, store in a brown bottle at 4°C in a refrigerator.

[0030] 2) Preparation of goat anti-bovine antibody nano-colloidal gold markers: take the required amount of nano-colloidal gold solution, and use 0.1 mol / L K 2 C0 3 (purchased from Sinopharm Chemical Reagent Co., Ltd.) to adjust the pH value to 7.6. Then, 2.5 μg of goat anti-bovine antibody (purc...

Embodiment 1

[0034] The symmetrical positions on the NC membrane in the round hole of the reaction box prepared in the above 3 are as follows: one point is IBR antigen ((0.95-1.81) mg / mL) 1 μL as the detection point, and the other point is Staphylococcus protein A (purchased in Ministry of Health (Shanghai Institute of Biological Products) (1g / L) 1μL as a quality control point; add 100μL blocking solution after drying at room temperature; after the blocking solution is dry, add serum to be tested (50-100) μL; add washing solution 100μL after infiltration Wash, repeat 2-3 times; add 100 μL of goat anti-bovine antibody colloidal gold marker; add 100 μL of washing solution after infiltration, repeat 2-3 times, wash away unbound goat anti-bovine antibody colloidal gold marker. If red dots appear on the membrane within 5 minutes, it is positive, and if no red dots appear, it is negative. As a sign that the test is valid, red spots should appear at the quality control point, otherwise the test i...

Embodiment 2

[0036] The selection of the optimal antigen concentration and the optimal concentration of goat anti-bovine antibody colloidal gold markers were purified with 5 concentrations of 1.81 mg / mL, 0.95 mg / mL, 0.4525 mg / mL, 0.2263 mg / mL and 0.1131 mg / mL respectively Spotting 1 μL of IBR antigen, repeated 4 times, added 100 μL of clinically confirmed critical IBR positive serum by ELISA method, and added different concentrations of gold-labeled goat anti-bovine antibody after washing (1 / 40, 1 / 20 of the original volume, respectively) , 1 / 10, 1 / 5), for cross-reaction. Screen the optimal antigen concentration and the optimal goat anti-bovine antibody colloidal gold marker concentration. Such as figure 1 As shown, the experiments done in this example are as follows: 1 μL of IBR antigen was prepared and coated in parallel and symmetrically on the inner circular holes of reaction boxes A to D, and the concentrations of 1 to 5 were 0.1131 mg / mL, 0.2263 mg / mL, and 0.4525 mg / mL, respectively....

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Abstract

The invention discloses a dot immunogold filter kit for detecting an IBR (infectious bovine rhinotracheitis) virus antibody and a detection method thereof. The kit comprises a) an infectious bovine rhinotracheitis virus antigen, b) a gold marked goat anti-bovine antibody, c) a cleaning solution, and d) a confining liquid. The method comprises the following steps: dotting the infectious bovine rhinotracheitis virus antigen on a nitrocellulose film; closing, and adding a serum sample to be detected; cleaning, and detecting the infectious bovine rhinotracheitis virus antibody by using the gold marked goat anti-bovine antibody as colloidal gold marked protein. Detection of the infectious bovine rhinotracheitis virus antibody by adopting the kit disclosed by the invention has the advantages of specificity, sensitivity, quickness, reliability, intuitive effect, easily determined result and the like, special equipment is not required, and the detection result can be preserved for inspection.

Description

technical field [0001] The invention relates to a gold-labeled immunodiafiltration kit and a detection method for detecting IBR virus antibodies. Background technique [0002] Infectious bovine rhinotracheitis (IBR) is a contact infectious disease of cattle caused by bovine herpesvirus type Ⅰ, characterized by high fever, dyspnea, inflammation of upper respiratory tract and tracheal mucosa, reproductive tract infection, Inflammation is the main feature. Generally, the clinical incidence rate of cattle is about 20%-30%, but the seropositive rate is much higher. Because the disease has the nature of latent infection and long-term detoxification of the body, antibody-positive cattle are actually recessive carriers. IBRV has typical pantropicity, can invade various organs and tissues, cause various clinical symptoms, and cause large economic losses to the cattle industry. It is one of the animal diseases that importing countries should pay close attention to, and the Ministry o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/531
Inventor 王武军徐淑菲孔繁德白泉阳唐泰山高丽钦刘正才黄一帆郑腾张体银
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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