Zoom Plate Assay Protocol - Bead-Based Fluorescent Sandwich Assay

Reagents:

5 µm diameter silica beads conjugated with capture antibody, 1% solid stored in 1% BSA-PBST
Antigen standard
Detection antibody conjugated with Alexa Fluor 647, 100 nM in 1% BSA-PBST
Blocking buffer: 10% BSA in PBS
Rinsing buffer: 1% BSA-PBST

Procedure:

Step	Procedure	Time	Note
1	Bead mixture incubation	30min	Each bead mixture include 10 µl beads, 10 µl antigen standard, and 1 µl detection antibody
2	Blocking with 50 µl blocking buffer	20min	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. Make sure all solutions get absorbed completely. If there is a bubble trapped at the reaction zone, use a 20 µl pipette to suck it out. Do not wait for more than 40 min, otherwise the membrane will get dried.
3	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.
4	Rinse with 50 µl rinsing buffer for 4 times	0	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. Make sure all solutions get absorbed completely. If there is a bubble trapped at the reaction zone, use a 20 µl pipette to suck it out. Optional: you can rinse with 40 µl rinsing buffer for 5 times. If using a robot, try continuous dripping a total of 200 µl rinsing buffer. The plate is now ready for fluorescent signal detection.