I am using an ELISA kit to measure APRIL in human serum. This kit provides a liofilyzed standard that we have to reconstitute and make dilutions in the plate. The curve is supposed to go from 0.78 ng/ml to 50 ng/ml.
The problem is my curves are not so good. I have the OD values obtained for the QC of this kit lot by the kit company and the values I am getting are much lower, especially in the least concentrated points (see attached table).The kit says to wait 10 minutes after pipetting the TMB substrate and observe the color to decide when o add the stop solution. In my first test I waited for 15 minutes, but it sure wasn't enough. In the second test I waited for 30 minutes, and the values are still low (visually, it seemed not to be changing for the last 10 minutes or so).
Besides this problem, I am not having good duplicates, even for the curve (it is worst for samples). And the shape of the curve is also not so good!
One possible critical point is that we do the washes manually. What I do is to use a multichannel pipette to fill the wells (I pipette 190ul twice on each well), then I use the same pipette to take off half the volume and then I turn the plate upside down to get rid of the rest of the volume (using a paper towel to dry the plate before turning it up again). I tried once not to aspirate half the volume, and directly turn the plate upside down, but the result was terrible. Maybe I am using too much or too little strength when turning the plate. Or maybe I am taking too long to pipette twice and then aspirate half the volume before turning the plate (the kit protocol says we should wait about 10-15 seconds before emptying the wells).
What do you think is the issue here? Can it be solved?
Just in case someone wants to know, the kit is this: http://www.ebioscience.com/human-april-platinum-elisa-kit.htm
I attached the results for my curves.
Thanks in advance for the help!
Did you try use plates from other company? Maybe the coating step isn't efficient and the amount of bonded antibody may not be enough to lead to a strong signal. We once used a same ELISA kit with plates from different companies and results were indeed different (sadly, the cheaper one was the worst).
If the problem was inefficient washing I think you would get a higher signal (and no different signal between concentrations) instead of low signal
I used Elisa kit same company with you. I dont think your std curve is strange. Please check the file to calculate your std curve as attached file that I got from this company. It seem your result is still ok. But absorbance is weak. So I recommend
1. After added TMB Substrate Solution , you should keep at dark place, and should not wait too long. I think 10 mins that is enough. After added stop solution, should measure immediately. This reaction is very quick that after reaction happened, it will decrease .
2. should wash around 1 min per once time from this company ELISA protocol
3. after washing, please check wells that dont have washing buffer
4. should dilute std in another well, and add once time to wells that you will measure
5.I did not used multiple pipette to add std too, but should quickly add
6. After added solution in every step, please shake plate weakly around 10 sec
I hope that it will improve your results
GOOD LUCK
Your data does not look as horrible as you describe. CVs of 5% and sometimes more are quite common in ELISAs. The ODs in the first data set are a bit low, maybe a dilution mistake. (if that happens repeatedly, increase the concentration of the secondary AB slightly might help).
About the washing: Add 200ul of PBST per well with a multichannel pipette or a stepper and then grab the plate, throw the liquid into the sink and beat the plate 3 times fiercely onto a stack of kitchen towels or similar to remove residual liquid. Repeat the procedure 2 times. Same procedure applies to the removal of coating liquids etc. Beats every microplate washer, but makes you crazy when doing stacks of plates :).
However, I noticed that in the second data column from each of your experiments, almost all data points have smaller values than in the first column. This might indicate a problem with the general setup (maybe pipetting). Can you grab someone in the lab next door who is considered an "ELISA expert" and might want to supervise you doing an experiment?
In the kits I'm using the antibodies already come in the plate, no way of changing it.
Apparently my data isn't as bad as I thought it was, guess I was being too strict.
Anyway, thanks for the tips. I'll try to dilute the standard out of the plate and then transfer it. And thank for the files to the curve calculations.
This tip on the wash is also very useful, maybe I'll star adding less buffer, around 200ul instead f the 400ul I was using, then I can directly throw the liquid as you said and don't need to aspirate some of the liquid before...
Thanks again for the help! :)
It's my pleasure.
In addition, protocol recommend wash buffer >250. Now I use 250 ul per 1 time. So you also can add just 1 time.
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