Negative concentration??
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 Joined: Wed Oct 21, 2009 5:19 pm
Negative concentration??
I carried out an ELISA to determine the concentration of TNFalpha in 6 different samples. A standard curve was created in Excel and by fitting the trend line, the equation of the graph was determined to solve for the concentration of TNFalpha. However, when working out the concentration of TNFalpha most of the values were fairly close to where I estimated they should be except the very last concentration which when worked was a negative concentration. How is this possible? Also the xaxis has a logarithmic scale but the trend line is linear with the equation below. Any help greatly appreciated!!
Sample Mean Absorbance at 450nm
1 0.821
2 0.752
3 0.424
4 0.445
5 0.433
6 0.054
Equation of graph y=0.0089x + 0.1235 (where y=mean absorbance at 450nm and x=concentration TNFalpha (ng/ml))
Sample Mean Absorbance at 450nm
1 0.821
2 0.752
3 0.424
4 0.445
5 0.433
6 0.054
Equation of graph y=0.0089x + 0.1235 (where y=mean absorbance at 450nm and x=concentration TNFalpha (ng/ml))
You can see, that theoretically, if your concentration was zero (x = 0 ng/ml), than you wil get absorbance of 0.1235. That means, that you have bad baseline (that is, your blank sample is actually not blank, but absorbs), but more likely Excel just found the best fit, which just starts a little bit above zero So, you can choose to push the line to zero (so you will get something like y = a.x) and see, how much different it will be, or you will just assume, that your last sample contains no TNFa
http://www.biolib.cz/en/main/
Cis or trans? That's what matters.
Cis or trans? That's what matters.
Re: Negative concentration??
freeradical wrote:Also the xaxis has a logarithmic scale but the trend line is linear with the equation below.
Regarding this. It's your choice, what type of trend line you like (or better, chooce). You should see the R^2 value and also see, what is reasonable (like get something like y = a.x^4 + b.x^3 + c.x^2 + d.x + e doesn't make much biological sense ). So, try both linear and logarithmic and you will see, what fits better
http://www.biolib.cz/en/main/
Cis or trans? That's what matters.
Cis or trans? That's what matters.
You shouldn't be trying to see what fits better. The model you use should reflect the physical of the system, i.e. light absorbance is linear and not quadratic or logarithmic no matter how good the line fits.
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Enjoying one moment at a time;
Accepting hardships as the pathway to peace;
~Niebuhr
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