Indirect ELISA Protocol
Indirect ELISA diagram courtesy of Univ. of Florida. Click to enlarge.
- 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.
1.16g Na2HPO4, 0.1g KCl, 0.1g K3PO4, 4.0g NaCl (500ml distilled water) pH 7.4.
- Blocking solution:
Common blocking agents; 1% BSA, serum, non-fat dry milk, casein, gelatin in PBS.
- Wash solution:
PBS or Tris-buffered saline (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).
- Antibody dilution buffer:
Dilute primary and secondary antibody in 1x blocking solution to reduce non-specific binding.
- 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. Seal the plate and incubate at 4℃ overnight or at room temp for 2 hours.
- Wash plate 3 times with PBS.
- Add 200µl blocking buffer, 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 PBS.
- Add 100µl of diluted primary antibody to each well.
- Cover the plate with an adhesive plastic and incubate for 2 hours at room temperature.
- Wash the plate 4 times with PBS.
- Dilute 100µl of conjugated secondary antibody to the optimal concentration (according to datasheet) and then add 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 PBS.
- Using a multichannel pipette, dispense 100µl (or 50µl) of the substrate solution into each well.
- After sufficient color development, add 50-100µl of stop solution to the wells if necessary.
- Record the absorbance at 450nm 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