I won't be able to finish processing my ELISA, and I wanted to know if it would be possible to keep the secondary coated overnight, or if the whole thing is just useless now.
Hello Ghadeer Alhamar
Incubation with secondary antibody should not be kept for long in ELISA. Longer exposure can lead to high background. Usually the incubation time for the secondary antibody in ELISA is 1-2 hours.
Best Wishes.
I agree with Malcolm. The elisa is optimized for specific time points and assay conditions. the background will be higher . You may gleen qualitative insight at higher analyte concentrations but the quantitative element and the ability to detect low analyte levels is essentially lost
2 hours of RT incubation is almost equivalent to overnight incubation at 4C. So, if you donut have an option you better incubate the secondary overnight at 4. Worst case you will notice an increase in background. Good luck.
As just stated the equivalence of room temperature (or even 37c) for 2-3 hr or overnight at in the fridge at 4-8c is fairly standard/accepted. I would not think it would make any difference re background. There are many other factors in the assay steps that are much more likely to influence this eg use and type of blockers to non specific binding, dilutions of reagents used etc
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