I am performing a Sandwich ELISA using a fluorescent-labelled detection antibody which eliminates the need for a detection substrate. The basic steps involved in my assay are: capture antibody immobilization, washing, blocking, washing, sample & standard incubation, washing, and incubation with Alexa488-conjugated primary antibody. After the recommended incubation time, I read the fluorescent signals without washing the wells. Fluorescent readings were similar for all standards. I have repeated the experiment several times with different antibody dilutions, blocking solutions, and incubation times, however fluorescent signals remain similar for all standards. Is there anything I am not doing right? Do I need to wash the wells before using the plate reader?
Yes, you need to wash out the unbound fluorescent antibody before reading the plate.
Yes, you should wash before reading the plate to avoid the unreliable results.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence