I am trying to optimize the ELISA assay for human ODC. I did the triter plate (4 different concentration of lysite, 3 different dilution of antibody, I used also b-actin as a reference). I constantly get the high background with ODC antibody, with b-actin I don't have this problem. I am using the 5% skimmed milk for blocking solution, 1xPBS 0.2% Tween for washing (3-4 times) solution, I diluted antibody 1xPBS (after adding the 1% of milk I din't get any signals, with BSA it was working for the first time, but today I didn't get any signal). I've tried extending the blocking step an also I did the higher concentration of blocking solution it is still the same. Does anyone have any idea what could be the reason?
Yes I will change the blocking solution for 0.5-1% BSA. For washing I used the 0.2% PBS Tween, and changing the number of washing didn't give any results. I am doing the indirect ELISA. First I coating plates with cell lysate, after that is blocking step, primary antibody (a try optimize the dilution) and secondary antibody conjugated with HRP (1:10000).
With primary antibody 2h 37C, secondary 1h 37C. Thanks Dewald for all advice.
Hi, Bruno the ODC antibody was checked in Western Blott and din't give any crossreactivity. Which ODC antibody you used?
Dewald I've changed the incubation periods and it wasn't helpful (I was trying to do that in RT, it is not working). Based on the controls the secondary antibody don't produce any background. Today I've tried to increase the volume of the blocking solution (from 200ul to 300 ul per well), because maybe something binds also to the wall of the wells but still now improvement. I prepare all reagents in the mornig in sterile conditions.
Dear Magdalena, in my experience, ODC Western blot was disheartening. So, I am not surprised of your difficulties with the antibodies. My advice is to send your samples to an external lab and discuss, step -by- step, the procedure with the colleagues, or better, to perform your assay in a host lab, if you do not want to waste both your time and patience.
Hi guys,
Small update, the ELISA isn't working still after changing the blocking solution, so only one thing left. I am ordering new antibody. I was using this sigma one and some time ago other person used that and it was working, so it is possible that antibody degrade ted. I was store in aliquots in -20C, so should last for long time. But it was transported from other place and might defrosted during the transport and be frozen again. And as you know antibody doesn't like that. So I hope so the new antibody will be working.
Thanks for all your help.
Luciano I am not give up so easily :).
Hello Magdalena,
It's been a while since your original post. Did your experiment work in the end?
Thank you,
Ricardo
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA