Zoom Plate Assay Protocol - Bead-Based Chemiluminescent Sandwich Assay
- 5 µm diameter silica beads conjugated with capture antibody, 1% solid stored in 1% BSA-PBST
- Antigen standard
- Detection antibody conjugated with HRP or AP, 10 nM in 1% BSA-PBST
- Blocking buffer: 10% BSA in PBS
- Rinsing buffer: 1% BSA-PBST
- Enhanced Chemiluminescent (ECL) substrate
cat# BGF27B (Glassfiber 2.7 µm pore size, black well, for bead assay)
Bead mixture incubation
|30min||Each bead mixture include 10 µl beads, 10 µl antigen standard, and 1 µl detection antibody|
Blocking with 50 µl blocking buffer
This step can be done concurrently with Step 1.
Inject at the center of the reaction zone. Pipette tip should be close to the membrane but do not touch the membrane.
Add 20 µl of bead mixture onto the membrane
|0||Inject at the center of the reaction zone. Pipette tip should be close to the membrane but do not touch the membrane.
For higher sensitivity, you can add 20 µl of rinsing buffer onto the membrane before adding the primary antibody to reduce background.
Rinse with 50 µl rinsing buffer for 4 times
When injecting, rest the pipette tip against the wall near the reaction zone. Do not pipette over the reaction zone as this is easy to introduce bubble.
Add 10 µl ECL substrate
Inject ECL substrate solution toward the center of the reaction zone. Pipette tip should be close to the membrane but do not touch the membrane. Injection to the wall will result in reduced signal level.
Incubate for 0-10 min and read luminescent signal from a gel imager or plate reader.
You can rinse away the previous ECL by adding 20 µl rinsing buffer and add additional 10 µl ECL substrate for multiple readings.