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ELISA assay in triplicate


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4 replies to this topic

#1 ari_doodle

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Posted 11 February 2009 - 11:53 AM

I have been working with a commercially available ELISA kit that is known to have some problems. I do the samples in triplicate and it seems that when I look at the data there is one of the three results that does not agree with the other two. Can I disregard this data point?

#2 tonix37

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Posted 12 February 2009 - 02:03 AM

I have been working with a commercially available ELISA kit that is known to have some problems. I do the samples in triplicate and it seems that when I look at the data there is one of the three results that does not agree with the other two. Can I disregard this data point?



Is possible that you have an edge effect between triplicates. This can be solved incubating your ELISA plates in an approppiate moist chamber.

You can read the Edge Well Effects from the link ELISA guide.
This phenomenon refers to the observation that occasionally ELISA results show a variance in their ODs between edge
wells and the wells in the central region of the plate. In the past this has been attributed to manufacturing variations in
plates leading to higher adsorption in the outer wells. More recently manufacturers have begun demonstrating and certifying
the consistency of the manufacturing process by testing and reporting the well-to-well variation of replicates
performed in all wells of a plate. For instance, Nunc certified plates are guaranteed to have a CV in replicate wells of <5%
and no wells with >10% variation.

#3 scolix

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Posted 12 February 2009 - 05:16 AM

At times when one of the triplicate sample gives completely weird results, throw it off. You will have a weird one every now and then. But I would normally repeat the ELISA again just to make sure the effect or whatever you see in the first ELISA is reproducible.

cheers

#4 Gerard

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Posted 12 February 2009 - 07:02 AM

Can I disregard this data point?


Your setup is to do samples in triplicate, then use the results unless there is a good specific reason to disregard a datapoint, just a difference in the results is not enough.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#5 molgen

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Posted 12 February 2009 - 07:24 AM

I don't feel comfortable with discarding 1 out of 3.
Not enough repetitions in my mind.




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