Direct ELISA Protocol

Direct ELISA Protocol

Direct ELISA diagram courtesy of Univ. of Florida.

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. Dilute the antigen to a final concentration of 10µg/ml in bicarbonate/carbonate coating buffer. Pipette 100µl of the antigen dilution into a PVC Microtiter Plate to coat the wells. Dilute down the plate as necessary with serial dilutions of the antigen. Seal the plate with an adhesive plastic and incubate at 4℃ overnight or at room temp for 2 hours.
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 plate with an adhesive plastic and incubate for at least 2 hours at room temperature or overnight at 4℃.
5. Wash the plate twice with wash solution.
6. Add 100µl of the antibody diluted to the optimal concentration (according to manufacturer datasheet) and then dilute with blocking buffer immediately before use.
7. Cover the plate with an adhesive plastic and incubate for 2 hours at room temperature.
8. Wash the plate 5 times with wash solution.
9. Using a multichannel pipette, dispense 50-100µl of the substrate solution per well.
10. After sufficient color development, add 50-100µl of stop solution to the wells.

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