The principle of enzyme-linked immunoelectrodiffusion measurement technology The principle of enzyme-linked immunoelectrodiffusion measurement technology is a new technology that combines immune electrodiffusion (Immunoelectrodiffusion, IED for short) and immunoenzymatic technology. The antigen and the IgG corresponding to the antigen quickly form an immune complex, and then the enzyme-labeled anti-IgG immunoglobulin is incubated with the immune complex, and then developed with a substrate. Enzyme-linked immunoelectron diffusion technology increases the sensitivity of immune electrodiffusion and is useful for analyzing complex antigen reactions and different types of immunoglobulins. Procedure equipment and equipment for ELISA technique (including electrophoresis tank); cellulose acetate film (2.5cm × 17cm); microliter pipette; large glassware and Other experimental glassware. Material and reagent soluble antigen solution; rabbit antiserum corresponding to the antigen; HRP-labeled anti-rabbit IgG antibody, in which the concentration of specific antibody protein is 1.25 mg / ml; 0.1 mol / L pH 8.2 barbiturate buffer; 0.2% Haemosol solution; 0.1mol / L Ph7.6Tris buffer; 0.01mol / L pH7.2 PBS; washing solution (85gNaCI, 500ml barbiturate buffer, distilled water added to 1000ml); substrate and hydrogen donor solution (H2O2 and DAB ï¼4HC1). Experimental procedure (1) Use soft pencil to draw dots on the cellulose acetate film. Each dot is 3cm away from the edge of the film and the opposite distance is 11cm. Mark the dots with a soft pencil in the middle of each dot. ⑵ Soak in 0.1mol / L pH8.2 barbiturate buffer solution, drip water, but keep it moist. (3) Spot using a microliter pipette, add 3ul (or 1ul) of the antigen at one point, and 15ul (or 5ul) of the rabbit serum against the antigen to be tested at the other point. Bridge with wet filter paper, the electrophoresis buffer is 0.1mol / L pH8.2 barbiturate buffer, the current intensity is 1mA / cm, the electrophoresis time is 2h, and the antigen end is connected to the negative electrode. ⑸ After electrophoresis is completed, immerse the membrane in the washing solution for 30 min to remove excess serum protein and free antigen. ⑹ Immerse the film in 0.01mol / L pH 7.2PBS, wash with shaking for 30min, remove the film, and drip the remaining water. ⑺Dilute the enzyme-labeled antibody 1:50. â‘» Incubate with diluted enzyme-labeled antibody at room temperature for 2h. ⑼ Shake and wash in 0.2% Haemosol solution for 15min. ⑽ Shake and wash in Tris buffer for 30min. ⑾ After immersing in substrate solution for 1min, observe the color development result.
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