3 replies to this topic

[red_monkey]

Hi there,

Easiest if I explain what I've done first...

Cell culture
12 animals
Cells seeded to 24 well plates.
Treatment - added in 3 wells per treatment (i.e. 3 biological replicates?)
The contents of each well were then frozen prior to ELISA.

ELISA
I took 2 of the above replicates from each treatment group and added them to the 96 well plate for ELISA in duplicate (technical replicate?).

So in total I've got 4 replicates from the same animal and treatment group. Firstly, is this ok to do? and if so, how should I go about analysing and describing the data? Can I use all 4 together so long as there is no statistical variation between the two types. If not, which should I ideally use? I'm having a nightmare trying to find the correct answer through papers.

Many thanks

[Ben Lomond]

It is not acceptable to create a mean of the 4 wells as the variation within each pair of wells is derived from different sources.

Mean the duplicate ELISA results from each single sample.  The sample contents should be the same, so any variation is within the limits of the performance fo the ELISA (and you can exclude results based on some predefined acceptance criterion based on % difference if this ends up being done as a routine assay)

This means that you now have a single result derived from each well of the 24 well plate, so you have two results for each treatment group. The variation in these two results is due to biological/experimental variation before it gets to the ELISA plate.

I am assuming you will then use some regression approach to describe the data based upon concentration of the treatment, as n = 2 for each treatment is a low number of replicates

[red_monkey]

Thanks a lot. Is it equally acceptable to use repeats from the one sample as a measure of assay precision, rather than biological variation. or is that not the typical thing done for ELISA analysis?

[Ben Lomond]

Yes, the same sample run multiple times can be used to estimate precision, but this is typically done in multiple wells in each of several assays