Sandwich ELISA Protocol

Sandwich ELISA diagram courtesy of Univ. of Florida. Click to enlarge.

Materials

1. Bicarbonate/carbonate coating buffer (100mM)
   Dilute the antigen or antibody in coating buffer to immobilize them to the wells:
   3.03g Na₂CO₃, 6.0g NaHCO₃, 1000ml distilled water, pH 9.6.
2. PBS:
   1.16g Na₂HPO₄, 0.1g KCl, 0.1g K₃PO₄, 4.0g NaCl (500ml distilled water) pH 7.4.
3. Blocking solution:
   Common blocking agents; 1% BSA , serum, non-fat dry milk, casein, gelatin in PBS.
4. Wash solution:
   PBS or Tris-buffered saline (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).
5. Antibody dilution buffer:
   Dilute primary and secondary antibody in 1x blocking solution to reduce non-specific binding.

Method

1. Coat the wells of a PVC microtiter plate with the capture antibody at a concentration of 1-10µg/ml in carbonate/bicarbonate buffer (pH7.4). Seal the plate and incubate overnight at 4℃ or 2h at room temperature.
2. Wash plate 3 times with PBS.
3. Add 200µl blocking buffer, 5% non-fat dry milk/PBS, per well to block remaining protein binding sites.
4. Cover the plate with an adhesive plastic and incubate for at least 1-2h at room temperature or overnight at 4℃.
5. Add 100µl of appropriately diluted samples to each well. Compare signal of unknown samples against those of a standard curve for accurate quantitative results. Standards (duplicates or triplicates) and blank must be run with each plate to ensure accuracy. Incubate for 90 min at 37℃.
6. Wash the plate twice with PBS.
7. Add 100µl of diluted detection antibody to each well.
8. Cover the plate with an adhesive plastic and incubate for 2h at room temperature.
9. Wash the plate 4 times with PBS.
10. Dilute 100µl of secondary conjugated antibody to the optimal concentration (according to manufacturer datasheet) and then add blocking buffer immediately before use.
11. Cover the plate with an adhesive plastic and incubate for 1-2h at room temperature.
12. Wash the plate 5 times with PBS.
13. Using a multichannel pipette, dispense 100µl (or 50µl) of the substrate solution per well.
14. After sufficient color development add 50-100µl of stop solution to the wells if necessary.
15. Record the absorbance at 450 nm on a plate reader within 30min of stopping the reaction.