Hi, I'm running an antibody-dependent phagocytoisis assay, where we add serum to fluorescent antigen-coated beads, then donor neutrophils, and subsequently measure the amount of phagocytoisis as MFI (fluorescence) within our neutrophil population to give us a phagocytosis score.
As we run the assay on different days with different neutrophil donors we are also running a titration of a serum standard. I was wondering how best to utilise this standard curve?
One method we're using is like what you would do with an ELISA and interpolate the values of our diluted sample from the standard curve. But this is only giving me a somewhat arbitrary value of the dilution required (which I can convert to phagocytic units, but again this is just done arbitrarily). The other method I've tried is min max normalisation using the top and bottom of the standard curve values.
What I would ideally like is a method of normalisation that gives an output as phagocytic score, as this seems biologically most relevant. My background is not in maths/stats so I'm not sure if there's a formula I could use to do this or if the methods above are fine. Any input on this problem would be much appreciated.
Cheers,
Mari
Hi Mari,
I do not really understand what you mean. Do you want to linearize the curve ?
Do you want to get a phagocitic score from a certain dilution level ?
Hi Marc, sorry I should add that I uses a four parameter log transformation on my curve first. Then we get a phagocytic score (MFI) from the assay (samples are run at a set dilution e.g 1 in 320) so I just need to use the standard curve to ensure consistency. Yes one way is reading off and getting a dilution value, but ideally I would like some way of using the curve to keep my answer as a phagocytic score if that makes sense?
I expecte may be interested in z score that is called standard value.
Z=(observation - avarege) /standard deviation.
Standard scores
The raw scores (data) obtained as a result of tests and measurements do not help to infer the levels of individuals tested compared to the mean or comparison between the people themselves, if we know that individuals may be subjected to a set of tests (test battery) and each of these tests enjoy The unit of measurement is different from the other, some of which are measured by the minute and its parts as an expression of the unit of time, including what is measured in meters and its parts as an expression of the unit of distance, including what is measured in degrees or the number of times, and for the purpose of eliminating the units of measurement so that we can collect laboratory degrees and give a uniform degree In the tests we use the standard score.
For the purpose of extracting the standard score it is necessary to extract the mean and standard deviation for each test, through which the standard score can be extracted.
You can transform your variable to a new variable as said by Abbas Lafta Kneehr sir. You can also try BoxCox transformation for achieving the same.
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