Wednesday, May 05, 2004
Description Sandwich ELISA for SCCA1 and SCCA2 Procedure 1. Dilute affinity purified rabbit anti-SCCA 1:400 in PBS (appropriate dilution of this antibody needs to be determined following each batch affinity purification). Add 100 ml of diluted antibody to each well of a 96-well ELISA plate (use Probind from Becton Dickinson and Company, NJ). Incubate at room temperature for 1 h. 2. Wash plates 3X with PBS/0.1 % Tween. Incubate wells with PBS/0.1% Tween/1%BSA at room temperature for 1 h to "block" against non-specific protein adsorption. 3. Prepare 1 in 4 serial dilutions of GST/SCCA1 or GST/SCCA2 in the blocking solution as follows: 200, 50, 12.5, 3.12, 0.78 ng/ml. Prepare dilutions in eppendorf tubes, then transfer 100 ml/well (or 50 ml if the sample volume is of limited quantity) in duplicates onto the plate. Incubate at room temperature for 1 h. 4. Wash plates 3X with PBS/0.1 % Tween. 5. Dilute mouse monoclonal antibodies 8H11 (anti-SCCA1 Mab, mouse ascites)) 1 in 4000 or 10C12 (anti-SCCA2) 1 in 1000 in the blocking solution. Add 100 ml to each well and incubate at room temperature for 1 h. 6. Wash plates 3X with PBS/0.1 % Tween. 7. Dilute HRP-conjugated goat anti-mouse IgG at appropriate dilution in the blocking solution. Add 100 ml to each well and incubate at room temperature for 1 h. 8. Wash plates 3X with PBS/0.1 % Tween. 9. Dilute 1 tablet OPD substrate in 5 ml OPD diluent (Abbott). Add 50 ml to each well. Incubate until yellow color has developed in standards and stop the reaction with 250ml/well of 1.25 M H2SO4 before any color change occurs in "zero" antigen wells. 10. Read the absorbance at 492 nm. Plot SCCA1/SCCA2 concentrations against absorbance at 492 nm using the four parameter equation with Softmax software program (Molecular Devices). Recipes Supplies Tips (责任编辑:泉水) |