广州医科大学《临床免疫学检验》2.实验课-ELISA-Hepatitis B.pptVIP

1、Double antibody sandwich method detected antigen (two-steps method) 1、Double antibody sandwich method detected antigen (one-step method) 2、Double antigen sandwich method detected antibody (one-step method)  3、competitive inhibition method detected antibody 4、neutral competitive method detected antibody Material preparation For ELISA 2、 enzyme： HRP is commonly used to label antigen or antibody. 3、substance：（chromogenic agent ） Ingredient: liquid A: contain 0.5‰ H2O2 liquid B：contain 0.26‰ TMB Mix two liquid together and use. 4、cleaning solution ： Before using it needs dilute first. cleaning solution 1 cleaning solution+ water 20 5、 stop buffer: concentration of H2SO4 is less than 2mol/l. 6、 Control?set：positive control and negative control. 7、 Neutral reagent: only prepared in HBeAb detecting reagent set. 1、 quantitative analysis ： standard sample: Establish?an appropriate concentration gradient: 1:2，1:22，1:23，1:24…….2n 2、Adding neutral antigen: Only needed to detect HBeAb, 50μL /?hole 3、 Adding enzyme-labelled antibody: 50μL /?hole 4、Incubating: mix together, cover the plate with sticky paper, 37?℃ for 30min. 5、Washing: abandon the liquid. Inject cleaning solution to each hole then shake plate gently for 1 minute. Abandon the liquid. Repeat 5 times. (Note: the last time pat the plate and make sure the plate is dry ) 6、Adding substance: Add 50μL liquid A and 50μL liquid B. Mix together. 7、Incubating: cover the plate with sticky paper, 37?℃ for 10min. 8、Terminal reaction: Add stop buffer 50μL/ hole to terminate reaction. The color will change to yellow. Test the result in 10min. 9、Result judging: Observing by eyes: Colorimetry : The detection wavelength is 450nm.