very higher background in ELISA which was going on very well before - (Nov/07/2005 )
here are some details of my elisa
1. coating(primary AB, monoclonal) 4°C overnight
2. washing(Tween 20 0.05% PBS)
3. blocking(BSA3% in washing solution)1h
4. washing
5. loading standard and plasma, incubation 2 h
6. washing 5*
7. 2nd AB conjugated to HRP, incubation 45 min
8. washing 3*
9. TMB 25 min
10 stop using sulfric acid and read
this protocol worked fine before, but i got very higher background in the last 2 days.
the reason seems to be that I used some new buffers, but I re-prepared them again and it did not work either.
another reason, which is the least possible, is that I used the newly ordered primary AB. could it be possible that this new primary AB has direct interactions with 2nd AB??? otherwise, there is no more explainations at all...
please drop me your kind comments
thanks
I use Carbonate/Bicarbonate buffer pH9.6 as a coating buffer. I found out that if I just use PBS, there will be high background.
I hope this may help.
I hope this may help.
thanks. but I also used this coating buffer.
anyway, another person in our lab is preparing all solutions once again, and she will do an ELISA today. so, we will see what will happen.
thanks.
unfortunately, we got the same results again.
I found the Lot. No. of new primary is different from the old one. could that be problem?
I found the Lot. No. of new primary is different from the old one. could that be problem?
May be. Is your primary antibody polyclonal or monoclonal ?
Hi,
If it worked well before it means that you change something...
you can have a strong difference in the old versus new primary Ab.
I recommand you to try both in the same time (if you still have an aliquot of the old one).
every time that I change one of my Ab or important reagent I make comparative controls in the following experiment just to be sure...
what you can also do is to change all your solutions and do it again.
did you do all the controls?
- protein, + primary, + secondary
+ protein, - primary, +secondary
+ protein, + primary, - secondary
TMB alone?
what are your results.
when you say background, It's just background (for exemple + 0.4 DO units in all wells) or all the signal are strongly amplified (all the DO are x2) ?
in the last case, may be you removed azide from your binding or washing solution?
because azide inhibits HRP activity, so if you remove the azide, your HRP work muwh more and your system is totally modified.
Maxime
unfortunately, we got the same results again.
I found the Lot. No. of new primary is different from the old one. could that be problem?
May be. Is your primary antibody polyclonal or monoclonal ?
Thanks.
my primary AB is monoclonal.
If it worked well before it means that you change something...
you can have a strong difference in the old versus new primary Ab.
I recommand you to try both in the same time (if you still have an aliquot of the old one).
every time that I change one of my Ab or important reagent I make comparative controls in the following experiment just to be sure...
what you can also do is to change all your solutions and do it again.
did you do all the controls?
- protein, + primary, + secondary
+ protein, - primary, +secondary
+ protein, + primary, - secondary
TMB alone?
what are your results.
when you say background, It's just background (for exemple + 0.4 DO units in all wells) or all the signal are strongly amplified (all the DO are x2) ?
in the last case, may be you removed azide from your binding or washing solution?
because azide inhibits HRP activity, so if you remove the azide, your HRP work muwh more and your system is totally modified.
Maxime
Thanks for your suggestions.
unfortunately, I don't have an aliquot of old primary AB (it is really a good idea).
regarding the background, I got an OD value of 0.9 for no antigen control(blank), and values of standard curve range from 0.9 to 1.6. however, the blank was 0.05 and curves ranged from 0.07 to 1.8 before.
I will try the controls you recommended.
Thank you again for your help.
Hi
If the latest set of tests dont work. Try sourcing your water from somewhere else. It might be the water quality? If the antibodies you are using are from a reputable company it is a bit unlikely they are going to vary very much from batch to batch as they generally perform a batch release test for each lot, in house. Could be bacterial or fungal contamination though.
Good luck
Sparky
Hi,
If it worked well before it means that you change something...
you can have a strong difference in the old versus new primary Ab.
I recommand you to try both in the same time (if you still have an aliquot of the old one).
every time that I change one of my Ab or important reagent I make comparative controls in the following experiment just to be sure...
what you can also do is to change all your solutions and do it again.
did you do all the controls?
- protein, + primary, + secondary
+ protein, - primary, +secondary
+ protein, + primary, - secondary
TMB alone?
what are your results.
when you say background, It's just background (for exemple + 0.4 DO units in all wells) or all the signal are strongly amplified (all the DO are x2) ?
in the last case, may be you removed azide from your binding or washing solution?
because azide inhibits HRP activity, so if you remove the azide, your HRP work muwh more and your system is totally modified.
Maxime
Thanks for your suggestions.
unfortunately, I don't have an aliquot of old primary AB (it is really a good idea).
regarding the background, I got an OD value of 0.9 for no antigen control(blank), and values of standard curve range from 0.9 to 1.6. however, the blank was 0.05 and curves ranged from 0.07 to 1.8 before.
I will try the controls you recommended.
Thank you again for your help.
Thanks so much.
we really did lots of things to check reagents, but I am afraid probably I won't be able to handle it......