ELISA std. curv best fit

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sereen kh
sereen kh's picture
ELISA std. curv best fit

Help!  :(

I'm working on ELISA, using ELISA kit ready coated, ELISA reader with software.

I make dilution series to create standard curve (1000 pg/ml, 500, 250, 125, 62, 31, 15) but sometimes I apply in the reaction only 5 std.

the cytokine i'm working with has very low level in human serum, almost around the lowest std. (15 pg/ml)

I got std. curve with R^2 > 0.99 which is very good but still not enough, because I need almost perfect std. curve!
where R^2 = 0.999 or even 1.0, otherwise I'll get undetectable samples conc.! so I need to delete/ignore some std. point to reach 0.999 value,  see the attachment.

I'm trying to delete only one point as much as possible using the software, but some times deletion two points (out of 5) is necessary especially the higher std. values.

My question is, is it ok if I delete 1000, 500 std. points while the sample conc. range between  0 - 250 pg/ml ? so the must important thing to get perfect linear curve within this range?

I confused & begin to wonder what are the rules to accept results!
what is the minimum acceptable R^2 value?
 Is it acceptable to delete one or two std. points?  
How can I choose the best parameters for fair results?!
When should I repeat the duplicated samples? what is the acceptable range of  the mean?

Any help will be highly appreciated.

Thank you :)

sereen kh
sereen kh's picture
I published this topic again

I published this topic again in Antibody based Technology, I don't know how to delete this topic from here!