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ELISA cut-off a controversial issue - (Dec/02/2009 )

Dear members of this Forum,
I found in references the ELISA cut-off is frequently calculated based on results obtained from a population of otherwise normal subjects (“healthy controls”). However I’m studying a new autoantibody I don’t know yet who would be in the “negative” population, and who should be considered “positive”; furthermore all tested human sera appear to have an increased binding to a particular “antigenic” peptide but not the other peptides of a similar length that I have in hands.

I found some publications in which a cut-off is calculated by adding 2 or 3 SDs on top of the mean OD of the blanc, or antigen-negative wells, but my boss does not like that type of cut-off.

I will appreciate very much if you the ELISA gurus could tell me your opinions on this controversial issue.

Cheers,

-bachai-

You have to look to the purpose of the cut-off value because that will define the groups and the calculations.
The calculation of a cut-off is by adding 2 or 3 SDs on top of the mean OD of the blanc is meant to tell with a difined certainty that a sample is not negative, this means that if your peptide is present in every sample this is not the way to calculate a cut-off.

-Gerard-

Negative Population: those without the disease; no autoantibody,
Positive Population: those with the autoantibody.

It sounded as though you were unclear as to the source of your samples. Young healthy, no history (& family) history of autoimmune disease would be my negatives.

Those with the clinical picture would be my positives. Those with autoimmune disease may have a spectrum of self directed antibodies and show up as positive in your test.

Plot the distribution of signals v. clinical picture. If all samples have signal that is ok...what is the intensity of the signals?

-sgt4boston-

Thankyou Gerard and sqt4boston.

"You have to look to the purpose of the cut-off value because that will define the groups and the calculations.
The calculation of a cut-off is by adding 2 or 3 SDs on top of the mean OD of the blanc is meant to tell with a difined certainty that a sample is not negative, this means that if your peptide is present in every sample this is not the way to calculate a cut-off."

Do you mean that a sample is NOT negative if OD due to its binding to the antigenic peptide is higher than the the cut-off calculated from wells containing nonreactive peptides, or no peptide?

"Negative Population: those without the disease; no autoantibody,
Positive Population: those with the autoantibody. Young healthy, no history (& family) history of autoimmune disease would be my negatives."

I agree with you about seggregation of disease from non-disease. But what if autoantibodies are a sort of natural autoantibodies (of low affinity) present in non-disease conditions?

-bachai-

sgt4boston on Dec 4 2009, 10:26 AM said:

Negative Population: those without the disease; no autoantibody,
Positive Population: those with the autoantibody.

It sounded as though you were unclear as to the source of your samples. Young healthy, no history (& family) history of autoimmune disease would be my negatives.

Those with the clinical picture would be my positives. Those with autoimmune disease may have a spectrum of self directed antibodies and show up as positive in your test.

Plot the distribution of signals v. clinical picture. If all samples have signal that is ok...what is the intensity of the signals?


I am new to ELISA and I also have a similiar problem. I need to learn what an appropriate positive and negative control is. I have a diseased group with a history of infection and a control group with no history of that infection. I suspect that only a very few patients in the diseased group will have a deficiency of an antibody. If I use pooled serum from the patients and pooled serum from the control group as the positive and negative controls and then find that the OD values are very close to each other then what can
I do?

-Namalee-