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plotting ELISA results


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4 replies to this topic

#1 biology_06er

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Posted 25 November 2011 - 11:57 PM

Hi there,

I recently carried out my first ELISA, and to each well I added varying concentrations of my antibody to my protein. I started with a 1:200 dilution and then titrated 2 fold so ended up having 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600 and then added each conc. to a well, and read plate.

I got readings and now have to plot them on a graph..just wondering what do I write on the X-axis...Y would be absorbance and X would would be concentration of antibody but how do I write concentration in terms of an X-axis...hope that makes sense!?

Thanks,
biology_06er

#2 biology_06er

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Posted 26 November 2011 - 12:31 AM

Could I just plot it as 200, 400, 800 etc and write as a 1/serum dilution (x100) (I know there a various ways as google shows but just wondering is there a standard one that works/looks best)

#3 BioMiha

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Posted 26 November 2011 - 01:08 AM

There is no standard way. As long as it makes sense to people you intend to show your results to you can write whatever you want.

#4 biology_06er

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Posted 26 November 2011 - 02:03 AM

cool, thanks for the reply :)...one other question..when determining antibody titre, it says it is determined by the greatest concentration that gives a positive result. Soo in saying that, when I ran my elisa and added the substrate to my wells, most of them turned blue (the thing is I can't recall at what dilution it stopped going blue) but I have a copy of my absorbance readings..the 1:1600 diltion is 1.36 the 1:3200 is 1.12 the 1:6400 is 0.781 and the very last dilution of 1:204800 is 0.087 so my question is what is defined as postive (a reading over one?)

#5 Ben Lomond

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Posted 26 November 2011 - 06:54 AM

So, I am assuming that you are adding the antibody to an ELISA plate coated with the protein target and you are generating your signal with a labelled antibody specific for the species in which the measured antibody is generated.

If you have only one antibody sample, then the actual titre test is a little meaningless, because you have nothing to reference it against. However, if your intention is to use the antibody as a reagent in some sort of future assay format, then you simply use the dilution that works for you.

If you will be having many antibody samples (such as serum samples) and you intend to obtain titre test data from each of them, then you will have to do a little more work.

In terms of defining what represents a positive reading at whatever dilution is considered appropriate for you, it will be necessary to have data relating to variability within a panel of blank matrix lots (so from serum samples in a panel of non immunized animals). If at a 100,000 fold dilution, you have a mean OD of 0.125 units, with a SD of 0.025 units in these blank lots, then some would say that the positive cutpoint at 1 in 100,000 is the mean + 2 standard deviations ( or 0.175 units which approxmates to the 95 percentile) you could be more stringent and go 3 SDs depending on what is it that you want to achieve. You could also do this test at multiple dilutions.

In order to standardize your data from different lots, it is useful to have a positive control reference sample to which you compare all your lots of antibody.

In terms of curve fitting, I would recommend having 1/dilution on the x axis, and response (OD) on the Y axis, and simply connect the points by hand rather than try to fit some arbitrary computer generated line to the points if this is simply a one-off experiment. If you intend to be running many samples, you will need the appropriate software to generate a 4 parameter logistic fit (4PL) or a 5 PL fit if the curve is s shaped and run the appropriate qualification experiments to demonstrate that the curve fit model is appropriate.

Let us know a few more details on what it is you are intending to acheive and how you get on

Good luck!




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