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Inconsistent ELISA results


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14 replies to this topic

#1 kee

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Posted 17 June 2009 - 11:07 PM

Hi all, I am getting inconsistent results for repeating sandwich ELISA runs with the same sample. Two plates were run in parallel for twice (at different day) with the same sample, but all 4 results were different. The standard curve is fitting well with an R^2 of greater than 0.95. The OD reading is obviously higher at column 3, 4 and 5, 6 as compared to the other columns (7 - 12; column 1,2 are standard). What are the possible reasons? How to troubleshoot this?

Thanks

#2 kausikdatta

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Posted 20 June 2009 - 05:14 PM

In order to answer your question, it would help to know the reagents that you are using and the outline of the protocol. Inconsistent ELISA can occur because of many reasons. What kind of value differences are you getting? What is your background reading?

#3 Minnie Mouse

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Posted 21 June 2009 - 07:45 PM

you may try to

1. Calibrate the ELISA plate reader

2. use a very good multichannel pipette

I hope this may help.

#4 kee

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Posted 22 June 2009 - 01:39 AM

In order to answer your question, it would help to know the reagents that you are using and the outline of the protocol. Inconsistent ELISA can occur because of many reasons. What kind of value differences are you getting? What is your background reading?


Thanks for answering my question.

I am coating the plate with anti-mouse IgG from goat in carbonate/bicarbonate buffer, incubate for 2 h at 37 degree C. Wash 4x with NaCl/Tween 20 and block with BSA/Tween 20 overnight at 4 degree C. Standard mouse IgG and samples from cell culture supernatant were loaded after washing process as above. After incubating at 37 degree C for an hour and half, repeat the washing process. The capture Ab is anti-mouse IgG HRP from goat, diluted in PBS at 1:1000 and incubate at 32 degree C for 1 1/2 h. Wash plate as before, then load ABTS solution and incubate for 45 min at 35 degree C. Read at 405 nm.

Example of values after calculation: sample A = 0.472, 0.454, 0.362, 0.649 ug/ml; sample B = 0.749, 0.299, 1.165, 0.324 ug/ml. Is the backgroud reading here means OD reading of empty plate? If yes, it is about 0.040. If you mean blank or negative control (diluent without sample), the OD reading ranging from 0.37 - 0.54.

#5 kee

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Posted 22 June 2009 - 01:42 AM

you may try to

1. Calibrate the ELISA plate reader

2. use a very good multichannel pipette

I hope this may help.


Thanks, answering the question already help. :o

I have just calibrated the multichannel pipette, but it may not be a good one. Yes, calibrating the plate reader might be one of the step that I should try.

Thank you very much.

#6 Gerard

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Posted 22 June 2009 - 02:44 AM

Check your washing step, it's the most likely cause for the bad results if your pipetting is OK.
Let the wash solution soak for 30 seconds between the washes.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#7 kee

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Posted 23 June 2009 - 12:48 AM

Check your washing step, it's the most likely cause for the bad results if your pipetting is OK.
Let the wash solution soak for 30 seconds between the washes.


Yes, I had already added 30s soaking step into my washing and even turn the plate after 2 washes each time.

#8 sgt4boston

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Posted 23 June 2009 - 11:56 AM

Can someone comment on the negative control? To me it appears the signal is very high for just buffer alone (no binding). If the negative control is a true blank it should read close to the 0 standard of the assay.

I think residual conjugate is left in the wells. Make sure you blot...bang out the plate on papper towels between each wash (manual).

Your %CV is very high for your samples...are you getting high CV for duplicates of your dose response curve as well? YOu can easily check the pipettes with an analytical balance.

good luck

#9 kee

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Posted 23 June 2009 - 10:27 PM

Can someone comment on the negative control? To me it appears the signal is very high for just buffer alone (no binding). If the negative control is a true blank it should read close to the 0 standard of the assay.

I think residual conjugate is left in the wells. Make sure you blot...bang out the plate on papper towels between each wash (manual).

Your %CV is very high for your samples...are you getting high CV for duplicates of your dose response curve as well? YOu can easily check the pipettes with an analytical balance.

good luck


My negative control is the culture medium, not buffer. I have tested with PBS, basal medium and culture medium as the diluent. They gave different range of standard curve, so I chose the one close to my sample, which is culture medium.

I did tap the plate on paper towel after the washing step, as dry as I can ....

Currently, %CV of my standard curve is very big, ranging from 8 - 35. The standard is normally place at column 1 and 2. %CV of column 3 and 4 is lower, at the range of 5 - 15. The rest of the columns are well below 10.

The pipettes were either new or just came back from calibration.

#10 sgt4boston

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Posted 24 June 2009 - 11:52 AM

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?

#11 kee

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Posted 24 June 2009 - 10:33 PM

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?


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.

#12 almost a doctor

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Posted 25 June 2009 - 01:26 AM

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.

#13 kee

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Posted 26 June 2009 - 01:12 AM

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.


My secondary antibody conjugated to horseradish peroxidase and I'm using ABTS.

My washing buffer is NaCl (0.9%)/Tween 20(0.05%).

#14 almost a doctor

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Posted 26 June 2009 - 01:58 AM

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.


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.

#15 kee

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Posted 26 June 2009 - 06:42 PM

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.


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.




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