ELISA -- dilution effects

S Kang stan_kang at hotmail.com
Thu Mar 14 19:00:09 EST 2002

we've been using a commercial kit to determine TNF-a concentrations in
mouse plasma samples. we were concerned that there wouldn't be a
signal due to the small volume of sample we had available.

the kit instructions call for 50uL of ELISA diluent and 100uL of
either sample or standard.

because of our concerns we ended up adding 50uL of undiluted plasma
into the sample wells. the signal we got fell within the range of the
standard curve. however, i wonder now if we overestimate the actual
concentration by comparing 50uL of our sample to a standard curve that
was generated with 100uL volumes of standard.

there is a debate between myself and the PI about how to correct for
this. do we correct for this by dividing our determined concentrations
by 2? unfortunately the company's technical service line wasn't that
helpful. they seemed to think that as long as the signal fell within
the range of the curve that it didn't matter what volume one used! or
even that one diluted the sample with the standard/sample diluent.

this is a purely technical matter. but it's one that's had me stumped.
and naturally one that casts some doubt in our determinations. any
criticism or feedback would be appreciated.

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