I am using an ELISA kit to detect BAFF on human serum. The kit has a standard curve that goes from 0.31ng/ml to 20ng/ml. In the literature, I found no article that used the exact same kit, but found that, with the other kits used, the concentration observed in normal "healthy" control individuals is around 1ng/ml (mean of 1.35, median of 0.93 and goes from 0.2 to 4.8 in different articles). No one reported individuals "negative" for the molecule. However, in my tests I am getting very low reads for the samples, most below the lowest concentration point of the curve (0.31ng/ml). Also, the OD values for the lower part of my curve are also low. I have the OD values obtained for the QC done by the company for the kit lot I'm using, and my curves give OD values that are higher for the most concentrated points (should be 1.6, I get 2) and lower for the least concentrated points (should be 0.15, I get 0.09).
I already tried adding more serum (no dilution, the kit says to dilute 1:3), or washing less times (the kit says to wash 6 times, I tried 4 and 5), but had no satisfactory results.
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, my serum samples are stored at -80°C and the kit is this: http://www.ebioscience.com/human-baff-instant-elisa-kit.htm
I attached the results for my plates, done with no dilution of the serum and 6 washes.
Thanks in advance for the help!
Hi Liana
Out of curiosity have you tested the standard curve solutions? Maybe try run your standard curve on a SDS PAGE gels to see if you can see your proteins, or maybe run the standards on a spec at 280 and calculate the concentration using the extinction coefficient and see if the concentration you get is lining up with the control. In general when I do ELISA I am quite rough with the washing steps. I remove most of the solution from the well with my multichannel and then I tap my plate several times on paper towel (I do this quite rough) to ensure all the solution is removed. I always do it 6 times if that is what the kit specifies. But if your primary concern with the assay is that the standard curve supplied is not giving you high enough absorbance readings try starting the trouble shooting with checking the samples. Remember as far as absorbance goes for a standard curve there is a limit.
Hi Liana
looks like a problem with the standards. According to the manufacturers homepage it is purified human BAFF fixed on the wells. Have contacted the company for support?Do they offer control samples?
The washing is seldom a problem, like Angela we are also quite rough during the washing procedure and the binding of BAFF to the antibodies and the binding of the antibodies to the wells shouldn´t be affected by this.
Do you stop the development of the colour in time? A prolonged step is sometimes the reason for too high values for the higher concentrations.
I am surprised that the company recommends dilution of the samples although the average concentration is in the lower range of the standards. Seems to be that there exist interferences with other substances in the serum which might disturb the ELISA.
Hi,
That's right, the standards for the curve come already fixed on the plate, along with many of the reagents, we just add water to the wells and incubate and then add TMB substrate.
I tried to talk to the company about it and they said it could be the washing steps. I'll ask if they may supply control samples it would be great! Thanks for the tip!
:)
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