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
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[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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