Recently I got a little bit confused. Generally, I was always doing blank correction to all my data in ELISA tests, but I started to wonder if I’m right in doing this because, in the case of diluted samples, it’s obvious that I should subtract blank (dilution buffer) readings, but what about undiluted samples? should I do it also with my samples which were not diluted? If so, why? They don’t contain this buffer anyway.
Swati, thanks for your answer..
Since the negative control is a solution without antibody or antigen, then yes, I may say that my blank serves as negative control... then, according to what you wrote I should subrtact it even from my undiluted samples, right?
And what about the case in which standards are diluted in water and samples in other buffer? shall I subtract water from standards, and buffer from samples?
Swati,
thank you very much for your reply. Usually it is as you wrote, standards and samples are diluted in the same buffer. But for example in this kit it’s different:
http://www.alpco.com/pdfs/30/30-6330.pdf
and producer is not writing how to do this calculations ( I guess I will ask them directly).
I agree with Swati, and even in the kit described above, the samples are diluted 1:200 or 1:5 in assaybuffer and measuring the signal from buffer-alone should suffice. If you would like to thoroughly check the background your assay provides, you could remove all antigen (albumin) from a stool and/or urine sample (by immune precipitation, for example), and measure this depleted sample according to protocol. The remaining signal is "background".
Performing such experiments can get you into a lot of work and validation experiments, and the added (clinical?) value of such procedures should be taken into account. As mentioned by Swati, substracting water-signal from your values will not substantially change your outcome measure, neither would substraction of the hypothesizes "background signal" above.
Keep it apart. A blank is necessary to evaluate the substrate reaction. Therefor should be nothing in the well. The negative control has no analyte. Thats to evaluate the background of the immune reactions in the assay.
For measurement you can measure either vs. the blank, than your calibration curve will be shifted (by the value of the negative control) upwards = intercept of the curve.
If you are measuring vs. the negative control, you will have no intercept, the curve will pass "0" at the OD-axis.
Why is it so important to have the blank: e.i. If your HRP-substrate is contaminated with some iron ions, this will catalyze the reaction as well. I'm all assays I have done, I used also a "full reaction control": adding substrate + enzyme. It hasn't to be on the plate. By this reaction you can see if the substrate is complete (including H2O2 for the HRP).
Wesley,
no no, I don’t want to check the background of my assay thoroughly, I just wanted to get the simple answer...
but, just in case one day I would like to do it, thank you for your suggestions :-)
Stephan,
as I wrote before - generally I was doing so called “blank correction” to all standards and samples. Usually “blank” it was a buffer in which I diluted standards and (if needed) samples – buffer with no antigen, so I may call this blank “negative control”. This blank (NC) wells I treated as the rest of the plate i.e. I was adding conjugate, substrate, washing buffer and stop, then I was subtracting blank’s OD from ALL wells (usually manufacturer suggest to do so).
I just started to wonder if this should be done also if my unknown samples are NOT diluted.
According to Swati’s answer, I should do this even then, do you agree with her?
Thank you Swati...
below is the answer from the producer:
"As blank we recommend the washing buffer, it will be diluted 1 : 10 with ultrapure water, thereby the quality of the water will also be tested.
Because by using the washing buffer the plate will be washed continually. Otherwise the standard buffer and dilution buffer are the same buffer."
You could continue to do blank subtract by substituting buffer for sample as others have recommended. However, as you have implied in your initial question, this would not be completely appropriate for undiluted samples. There is really no way to adequately account for background reactivity or actual inhibition of signal (inhibition of antigen binding to capture antibody) on undiluted samples unless you can have a true negative control by preparing something like Wesley Jongbloed suggested. The bottom line is the validity of performing this assay on undiluted samples is highly questionable. This is apparently a commercial kit for measuring albumin in biological fluids, and samples must be diluted as prescribed by the kit developers.
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