Sandwich ELISA for SCCA1 and SCCA2

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).

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Sandwich ELISA for SCCA1 and SCCA2