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.
Blocking solution: Common blocking agents; BSA, serum, non-fat dry milk, casein, gelatin in Block Ace.
Wash solution: PBS or TBS (pH 7.4) with detergent such as 0.05% (v/v) Tween20.
Antibody dilution buffer: Dilute primary and secondary antibody in 1x blocking solution to reduce non-specific binding.
Method
Dilute antigen to a final concentration of 1-20μg/mL using PBS or bicarbonate/carbonate coating buffer. Pipette 50µl 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 adhesive and incubate at 4℃ overnight or at room temp for 2 hours.
Wash plate 3 times with wash solution.
Add 200µl blocking buffer (e.g. 5% non-fat dry milk/PBS) per well to block remaining protein binding sites. Block ACE or BSA may also be used.
Cover plate with an adhesive plastic and incubate for at least 2 hours at room temperature or overnight at 4℃.
Wash the plate 3 times with wash solution.
Add 100µl of the antibody diluted to the optimal concentration (according to manufacturer datasheet) and then dilute with blocking buffer immediately before use
Cover the plate with an adhesive plastic and incubate for 2 hours at room temperature.
Wash the plate 4 times with wash solution.
Add 100µl of conjugated secondary antibody diluted to the optimal concentration (according to manufacturer datasheet). Dilute with blocking buffer immediately before use.
Cover the plate with an adhesive plastic and incubate for 1-2 hours at room temperature.
Wash the plate 5 times with wash solution.
Using a multichannel pipette, dispense 50-100µl of the substrate solution into each well.
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**