广州医科大学《临床免疫学检验》6.理论课ELISA-AFP.pptVIP

Enzyme： HRP is commonly used to label antigen or antibody. Substance：（TMB Color?Development?Solution） Ingredient: liquid A: contain 0.5‰ H2O2 liquid B：contain 0.26‰ TMB DH2 ＋ H2O2 D ＋ 2H2O Cleaning solution ： Cleaning sulution is the phosphate buffer with 0.05% Twain-20. Diluent: already prepared in reagent set. Stop buffer: the concentration of H2SO4 is 2mol/l. Control?set：prepared in the set. Double antibody sandwich method detected AFP (two-steps method) Preparation: Coating: antibody is diluted to the working?concentration and?added 50μL/ hole, incubated in 37?℃, 4h or 4?℃, 24h. Washing: abandon the liquid of the holes and wash with PBS?for 5 times,?every time 1?minutes. (in our experiment, the plates are already prepared for detecting ) 1、Adding sample: establish?an appropriate concentration gradient: 1:2, 1:4, 1:8, 1:16……(semi-quantitative) The diluted?samples?added to two or three holes so we can compare with result and reduce the error. 50μL /?hole. 2、incubating: put the plate at 37?℃, 20min . 3、washing: abandon the liquid of the holes and wash with PBS?for 5 times,?every time 1?minutes 4、Adding?enzyme labelled antibody : enzyme labelled antibody is diluted to the working concentration and?added 50μL/ hole 5、Incubating: put the plate at 37?℃, 20min. 6、Washing: discard the liquid of the holes and wash with PBS?for 5 times,?every time 1?minutes 7、Adding substance: liquid A (50μL/ hole) and liquid B (50μL/ hole) mixed together, 8、 Incubating: put the plate at 37?℃, in dark, 10min 9、terminal reaction: Each hole?adds the 50μL H2SO4 to terminate reaction. The blue color will change to yellow. Testing the result in 20min. 10、result judging: Observing by eyes: the deeper the color , the higher the concentration Testing by equipment: The detection wavelength is 450nm. Before testing the result, we