Background: I performed a DAS ELISA reaction at 405nm of 129 bacterial isolates (in addition to using controls) to identify the presence of my target bacterial strain. For my positive control, I received an OD value of 0.826, while my negative control received a value of 0.136. I obtained both of these controls from the ELISA kit, where the kit determined analytical data of control OD's to be:
>3.0 for positive control, 3.0. Could this be due to shortened incubation time? The protocol recommends 1-2hours of substrate incubation before beginning ELISA reading, however I incubated the plates for one hour and began the plate reading then. After incubation, I took readings every 5 minutes for a total of 40 minutes.
- These values which I listed were obtained 40 minutes after substrate incubation.
I would be thankful for any help! I hope the information I provided will be enough.
Hello, Peter!
ELISA's OD depends on the incubation time, if you incubated for 1 hour instead of 2 hours as recommended, you got a low OD value in the control as well as for the rest of the samples.
0.2 to 0.8 is considered the best OD, however, every kit have mentioned about it, which may be different than 0.2 to 0.8.
According to my experience, normally, in DAS-ELISA, unlike TAS-ELISA, there is a background (absorbance) in the plate, affecting the negative control value. Thus, check different steps which can be involved in this background to minimize it (e. g., washing steps, incubating in the wash buffer, the salt concentration of incubation/wash buffers, …). Besides, as you mentioned, better to go for more substrate incubation before beginning ELISA reading.
Also, for the next time, it is better to have several negative control (negative wells), and also your isolate obtained a value of 0.357 across different places of each plate as values might vary from well to well and even plate to plate.
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