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please find here below (in red) our customer's answer.
1. how long after purchase was the antibody used and found to give low signals?
The antibody was tested after less than one month storage in the fridge 8+4°C)
2. how was the substrate captured to the plate? Under what temperature, time?
Substrate was diluted in PBS 1x and coating was performed at 4°C overnight
3. how was the primary antibody incubated (temperature, time? with agitation?
the primary antibody was incubated at 37C for 1 hour, with no agitation , in an incubator
4. what wash steps were employed?
3 times with PBST (PBS 1x added with tween)
5. was there any sample concentration related increase in signal (ie increase with increasing concentration)? Could you please share the data obtained? This would greatly improve my understanding of the problem encountered.
Here following is an example of one elisa I made,; coating was performed using Betalactoglobulin + glucose incubated at high temperature for short time; the second with BSA glucose at 37C for 1 day, pH7.2 dialized. Reading was carried out at 495 nm.
6. which secondary antibody was used with the experiment (manufacturer and catalogue number would be most helpful).
Biorad a GAM-HRP diluted at 1:2500
Asked on Jan 26 2012
Thank you for providing those extra details. There are a few points which would warrant optimisation in order to obtain better results withthis antibody:
From the datasheet we recommend that this antibody be stored at -20°C on delivery. It may be thathaving stored it at +4°C may have reduced its activity.
2. Substrate coating
It may be that optimising the coating conditions would improve the signal observed. I would suggest trying incubation for2 hours at room temperature. I would also consider trying to use a carbonate buffer for coating, a recipe and description of use can be found here:
Unless already doing so I would suggest blocking for2 hours at room temperature.
4. Incubation of primary antibody
Incubation at 37oC is not suitable for many antibodies. In the optimisation stages I would suggest using 2 hours at room temperature, or 4oC overnight can often produce stronger signals. If these conditions result in an observed standard curve, incubation at 37oC could then be attempted.
5. Secondary antibody
I could not find the exact concentration or recommended dilution of the secondary antibody being used however this can be crucial in your experiment. We would typically recommend a dilution in the region of 1/4000-1/10,000. This may yield more specific results.
I understand that you have been trying to get this ELISA to work effectively for some time and the effort you have gone to. I am therefore willing to offer a replacement with the antibody you want (ab27684). If you would still like to proceed with this please do let me know and I will have it arranged. In order to do this I will send the replacement directly to you, could you therefore provide your shipping and billing address as well as your institutions VAT number.
Many thanks for your assistance in this matter.
Answered on Jan 26 2012