May I know what else can I do to prevent this from happening? I tried 3 times with the same Elisa and I always get a high background for all my standard, control and samples despite followed the protocols given.
Sura Zahim Hussein
Popular answer
You can prevent this problem by using reference wave length at 630 nm.
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Bhairavi Vajaria
Possible causes for higher background are:
- Insufficient washing or contamination in wash buffer. Increase your wash number and length of wash steps. Wash your plate properly and make sure there are no bubbles in plate. Blot final wash on paper towels. Prepare fresh buffer, read plate immediately after adding stop solution.
- Capture Ab can cross-react with other components in the system, like the detector Ab, and yield high background. Purification methods can be performed to increase to purity/quality of Ab, such as protein A, protein G, affinity purification via conjugated antigen.
- Increase dilution of secondary antibody. Higher concentration of secondary antibody may cause higher background.
- Some antibodies are very sensitive to pH. You can try to change pH of coating buffer.
- The substrate you're using also matters. Some substrates have very high sensitivity causing higher background.
- Incubation time of substrate may be too long, shorten the incubation time of substrate.
- Substrate may have been exposed to light prior to use.
- Rule out the chances of higher background by modifying one at a time from any of the above possibilities.
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Sura Zahim Hussein
You can prevent this problem by using reference wave length at 630 nm.
8 votes
6 thanks
Md. Golzar Hossain
May be due to insufficient washing... Wash at least 5 times after filling the well.
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