Inconsistent ELISA results - (Jun/18/2009 )
sgt4boston on Jun 25 2009, 03:52 AM said:
Ok...lets confirm you do wash several times between additions of sample, conjugate etc?
You blot out the wash between each and every wash step.
Your pipettes are calibrated. (including your multichannel pipette if you use one)?
Your %CV is very high for your standards OD values...and also for your samples.
Your sample values will be much closer if the CV of your curve is tighter....should be less than 10%.
Let us know if your samples fall within the linear range of the curve. And what the analytical range of the curve is (including the ODs for the 0 and highest point)?
You are running multiple assays...are you running frozen aliquotes of samples...are samples changing over time?
I noticed surfactant in the blocking solution...could the tween be interfering with bsa blocking the plastic?
Are you sealing your plates during those incubations? Are you mixing the plate before the incubation of reagents?
You blot out the wash between each and every wash step.
Your pipettes are calibrated. (including your multichannel pipette if you use one)?
Your %CV is very high for your standards OD values...and also for your samples.
Your sample values will be much closer if the CV of your curve is tighter....should be less than 10%.
Let us know if your samples fall within the linear range of the curve. And what the analytical range of the curve is (including the ODs for the 0 and highest point)?
You are running multiple assays...are you running frozen aliquotes of samples...are samples changing over time?
I noticed surfactant in the blocking solution...could the tween be interfering with bsa blocking the plastic?
Are you sealing your plates during those incubations? Are you mixing the plate before the incubation of reagents?
Yes, I wash 4x between each steps, including 30s soaking between washes and turn the plate after 2 washes. Then, blot on papers after washing step.
Yes. Pipette just calibrated (multichannel - 3 weeks ago, single channel - 2 months ago).
95% of the samples will fall within the linear range of the curve if dilute 2x, and all fall within the range if dilute 3x. Highest OD for standard is about 1.45 in average, lowest is 0.49 in average. NC (culture medium as diluent) normally give OD values of about 0.40 in average.
Yes, my samples are stored in -20 degree freezer, after thawing I will sonicate for 5s before dilution and loading the samples. Thawed sample will normally kept at 4 degree C for repeat assay within 5 days. I am not sure whether the samples changing over time, but they definitely are if I re-freezed them.
I don't know could it be tween 20 interfere with the bsa blocking, but I remember that once I had forgotten to add tween 20 and I couldn't get any results at all, all ODs were almost similar.
Yes, I seal my plate with parafilm, place them in a seal plastic bag with wet tissue. The plates were incubated in an orbital shaker, shaking at 100 rpm.
-kee-
What detection method are you using, what's your secondary antibody conjugated to, and what substrate do you use?
I think your media only OD are a bit high, I usually get this around 0.040 - 0.050 (same as a completely empty well). Do you wash just with water before adding your substrate?, detergents such as tween will interfere with your substrate and make results inconsistent.
-almost a doctor-
almost a doctor on Jun 25 2009, 05:26 PM said:
What detection method are you using, what's your secondary antibody conjugated to, and what substrate do you use?
I think your media only OD are a bit high, I usually get this around 0.040 - 0.050 (same as a completely empty well). Do you wash just with water before adding your substrate?, detergents such as tween will interfere with your substrate and make results inconsistent.
I think your media only OD are a bit high, I usually get this around 0.040 - 0.050 (same as a completely empty well). Do you wash just with water before adding your substrate?, detergents such as tween will interfere with your substrate and make results inconsistent.
My secondary antibody conjugated to horseradish peroxidase and I'm using ABTS.
My washing buffer is NaCl (0.9%)/Tween 20(0.05%).
-kee-
kee on Jun 26 2009, 10:12 AM said:
almost a doctor on Jun 25 2009, 05:26 PM said:
What detection method are you using, what's your secondary antibody conjugated to, and what substrate do you use?
I think your media only OD are a bit high, I usually get this around 0.040 - 0.050 (same as a completely empty well). Do you wash just with water before adding your substrate?, detergents such as tween will interfere with your substrate and make results inconsistent.
I think your media only OD are a bit high, I usually get this around 0.040 - 0.050 (same as a completely empty well). Do you wash just with water before adding your substrate?, detergents such as tween will interfere with your substrate and make results inconsistent.
My secondary antibody conjugated to horseradish peroxidase and I'm using ABTS.
My washing buffer is NaCl (0.9%)/Tween 20(0.05%).
I'll suggest that after your last wash with NaCl - Tween you do a couple of quick washes with dH2O. I dont know what method you use to wash your plates but here is what I do:
I use a big bucket (5L) filled with buffer (usually PBS or TBS containing Tween). I dip my plates one by one to fill the wells, leave to soak for ~30-60 sec, then empty and repeat (I do all this at the sink).
After conjugate incubation, I was my plates 3-4 times with buffer and then I quickly rinse all bubbles (detergent) with tap water (with the plate upside down), and then dip the plates in a bucket with dH2O and wash them 2-3 more times.
I've always been told detergents interfere, and have seen my plates become green all over with ABTS if I forgot the water wash. Is like with westerns you'll always do a last couple of just TBS washes.
Hope this helps.
-almost a doctor-
almost a doctor on Jun 26 2009, 05:58 PM said:
kee on Jun 26 2009, 10:12 AM said:
almost a doctor on Jun 25 2009, 05:26 PM said:
What detection method are you using, what's your secondary antibody conjugated to, and what substrate do you use?
I think your media only OD are a bit high, I usually get this around 0.040 - 0.050 (same as a completely empty well). Do you wash just with water before adding your substrate?, detergents such as tween will interfere with your substrate and make results inconsistent.
I think your media only OD are a bit high, I usually get this around 0.040 - 0.050 (same as a completely empty well). Do you wash just with water before adding your substrate?, detergents such as tween will interfere with your substrate and make results inconsistent.
My secondary antibody conjugated to horseradish peroxidase and I'm using ABTS.
My washing buffer is NaCl (0.9%)/Tween 20(0.05%).
I'll suggest that after your last wash with NaCl - Tween you do a couple of quick washes with dH2O. I dont know what method you use to wash your plates but here is what I do:
I use a big bucket (5L) filled with buffer (usually PBS or TBS containing Tween). I dip my plates one by one to fill the wells, leave to soak for ~30-60 sec, then empty and repeat (I do all this at the sink).
After conjugate incubation, I was my plates 3-4 times with buffer and then I quickly rinse all bubbles (detergent) with tap water (with the plate upside down), and then dip the plates in a bucket with dH2O and wash them 2-3 more times.
I've always been told detergents interfere, and have seen my plates become green all over with ABTS if I forgot the water wash. Is like with westerns you'll always do a last couple of just TBS washes.
Hope this helps.
I use plate washer to wash the plate. There was someone over here recommended me to use just squeeze bottle. It seems like your method even simpler.
Thank you very much, I will try this out in my next run.
-kee-