Interpreting ELISA results - (Aug/12/2005 )

I'm about to start running sandwich ELISA assays to detect my protein (PKC alpha) in treated cancer cell extracts. I've got a basic protocol as I'm having to make the asay from scratch, but only one person in the lab has had any previous experience with actually running ELISAs and he is on holiday for the next couple of weeks (typical).

I've read a lot of previous posts about doing ELISAs many of which mention high background. The problem is how do I know if I've got a high background? A search of the literature shows that other people have done PKC ELISA assays, but they use a kit which detects total PKC concentration and I need to look at individual isoforms.

I know how to get the results I've just got no idea how to verify them. I'm planning to make up a set of pure PKC alpha protein standards, but I'm lost when it comes to how to decide which dilutions to use, although I assume I'd start from maximal binding and work downwards.

A lot is talked about "curves" and I've got no clue what a curve for this protein should look like. Maths is really not my strong point as I'm very discalculaic (I still count on my fingers!) so if anyone has a simple explanation I'd be very greatful.

Thanks
Rosie

Dear Rosie,

I have met very few exceptionally math-minding scientist so do not fret about you finger counting.

Standards and standard curve: the range to use for the standard is determined empirically. Use the range that is published for total PKC and maybe make some lower concentrations as well.

For example (these are random numbers): if other people use 1 µg/ml to 100 µg/ml as their standard, then try 0.1 µg/ml to 100 µg/ml. Set one blank, then try 0.1, 0.5, 1, 5, 10, 50, 100 µg/ml.

Run your ELISA with just a range of standard concentratios and see what readings you get. If the readings range from the blank (maybe 0.1-0.2 absorbance units) to 1-2 units and when you graph them, you get a sigmoidal curve, then you are fine (see attached). If the readings are all low, then increase your standard concentrations. If your reading are all high, lower the concentration range.

NOTE: each plate reader will have an "upper" limit of detection. If you do not know what it is, keep you high values below 3 abs units. Above this "upper" value, the reader will not read accurately.

NOTE2: Graphing standard curve: using excel or available graphing program. X-axis = standard concentrations. Y-axis = absorbance readings. You can then plug in the absorbance value to determine what concentration of PKC your unknown sample contains.

NOTE3: For accuracy, prepare duplicates of the standards and samples.

Background: How do you know if background is a problem ... when your vehicle control or untreated sample gives absorbance values that sit in your standard range. This means that your ELISA is detected non-specific protein or your controls contain the protein of interest.

Hope this helps,

AussieUSA.

Thanks AussieUSA, this is really helpful!