I performed ELISA for liposomes and got negative values in untreated control and few treatment groups. But positive control, negative control and other treatment groups are all positive.
Note: I am not talking about the OD values but I converted OD into pg/ml and got the negative values.
Thanks in advance.
There can be many reasons. Your results depends on the type of ELISA (DAS-, TAS, PTA-ELISA) you are testing your samples/groups. The specificity of the antibodies you are using is also important. If they are very specific (monoclonal), they are usually less sensitive and you can get more negative results. On the other hand, if you have non specific antibodies, the sensitivity is increasing, but the specificity decreases. Time and temperature conditions of ELISA are also very important.
Every method has a detection limit and also a quantitation limit. When you get negative values (which have no biological sense) it just means that the levels in your samples are below this quantitation limit. Thats all you can say about hese samples, that their concentrations are lower than certain limit (you can even calculate these value). If you really need to measure these low concentrations, you have to improve the sensitivity of the method.
I have performed ELISA for mouse Tumor homegenate to evaluate IL-2 levels however i got negative results in standard OD for serial single or double concenntartion of standard,But it shows R value 0.99,do i consider the value or can exclude for Protein measurement in Graph Pad prism?
I have performed ELISA for mouse Tumor homegenate to evaluate IL-2 levels however i got negative results in standard OD for serial single or double concenntartion of standard after blank correction,But it shows R value 0.99,do i consider the value or can exclude for Protein measurement in Graph Pad prism?
I used high sensitivity Kit for the ELISA experiment
It doesnt make any sense to include negative values in your standard curve. I guess you have some positive values, in the highest stndard c oncentrationsyou could fit these data , and use the resulting curve to calculate the cnocentrationsi n your samples. Everything that is negative haves concentrations below your detection limit and is useless.
Hi everyone - I ran into a similar problem and was wondering if there is a mathematical way to correct for the negative values or is the only solution to repeat the ELISA?
I have to disagree with Gertrudis Rojas , it is posible to get meaningful negative results from an ELISA after extrapolating the pg/ml values from comparing the OD of the samples to the standard.
When you extrapolate data from a standard, you obtain an equation from your standard that connects OD values to pg/mL values. You then use to extrapolate OD values into the value of your standard (pg/mL). This equation normally looks like: y= m*x + c, where y is your OD value, m is the slope of the line, x is the pg/mL value and c is the constant that represent the detection threshold. However, deviation in your standard will cause the "c" to deviate from the detection threshold. This way, even though the absolute minimum value that your ELISA can detect is 0.05 (for example), you might get a c of 0.07. This will cause every value between the detection threshold and the C to come out negative, but it does not mean that they are UNDER the detection threshold. They are meaningful data.
An example of this happens often in my ELISA assays when i do a dose response treatment, and in the lower values i can observe they follow a perfect dose response patter but in negative values. For example i treat samples with decreasing concentrations of a drug and I get these levels of the cytokine: 5pg/ml, 1pg/ml, 0.2pg/ml, -0.1pg/ml, -0.5 pg/mL, -0.3pg/mL. The negative values are real, but the mathematical extrapolation process is imperfect and causes them to come out negative.
I have the same question as Amjad Ramahi , so I would appreciate any help with this issue
Possible that the values in your sample are below the detection limit.
Sorry Chhavi, as I explained in my previous reply, samples are NOT necessarily below the detection limit.
I solved this issue a couple of years ago but forgot about this post. Since it is still active, let me try to explain.
Often when you get negative values it can be due to samples being under detection threshold, but some other times the negative values are clearly following a pattern that indicates they are not just fabrications. In these cases they can show as negative fue to using the wrong mathematical extrapolation method for your standard.
In my previous reply I explained how to extrapolate values using a linear equation, but this is only useful for values in the linear range of the standard. If you have low values, they will not be in that range.
In those cases (in fact in most cases) it is better to use a hyperbolic equation to interpolate your data from the standard.
It is lengthy to explain how to interpolate using the hyperbolic equation, but luckily for us there is software like graphpad that does it for us. Just put your data in an XY table on graphpad and use the curve fitting function on your standard. Lastly select interpolate unknown with 95%CI and you'll see how your negative values are no longer negative, while the rest of your data remains consistent.
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