Sandwich ELISA Protocol

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

Materials

1. Bicarbonate/carbonate coating buffer (100mM)
   Dilute the antigen in coating buffer to immobilize them to the wells:
   3.03g Na₂CO₃, 6.0g NaHCO₃, 1000ml distilled water, pH 9.6.
2. Blocking solution:
   Common blocking agents; BSA, serum, non-fat dry milk, casein, gelatin in Block Ace.
3. Wash solution:
   PBS or TBS (pH 7.4) with detergent such as 0.05% (v/v) Tween20.
4. 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 bicarbonate /carbonate buffer. Seal the plate with an adhesive plastic and incubate overnight at 4℃ or 2 hours at room temperature.
2. Wash plate 3 times with wash solution.
3. Add 200µl blocking buffer (e.g. 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 1-2 hours 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 solution.
7. Add 100µl of diluted detection antibody to each well. Dilute to the optimal concentration (according to manufacturer datasheet) with blocking buffer immediately before use.
8. Cover the plate with an adhesive plastic and incubate for 2 hours at room temperature.
9. Wash the plate 4 times with solution.
10. Add 100µl of conjugated secondary antibody diluted to the optimal concentration (according to manufacturer datasheet). Dilute with blocking buffer immediately before use.
11. Cover the plate with an adhesive plastic and incubate for 1-2 hours at room temperature.
12. Wash the plate 5 times with solution.
13. Using a multichannel pipette, dispense 50-100µl of the substrate solution into each well.
14. After sufficient color development, add 50-100µl of stop solution to the wells.

Record the absorbance on a plate reader within 30 min of stopping the reaction

**This is a suggested protocol and should be adjusted by the user accordingly**


Available ELISA Protocols: Direct ELISA | Indirect ELISA | Sandwich ELISA