I am currently performing an ELISA test to investigate the result of immunization of surface proteins from a chicken parasite. The negative controls used during the immunization includes the unimmunized chickens, as well as the PBS controls. Hence for the calculation of the cut off value, is it the unimmunized chickens will be more suitable compared to PBS? Is that I should take the value of ELISA given by the serum of unimmunized chicken after the last boosting? (although theoretically there should be no large differences between the readings at any time point as the chickens are not injected with anything). Need help to clarify this and hope to have a clear definition of the 'cut-off value'' too. Thanks a lot.
Option 1: Pool equal volumes of the samples of _all_ the chickens prior to first immunization and use that as the negative control for establishing the cut-off value (if you have enough). The amount of total IgY in the un-immunized group might increase with age, leading to some low titers at the later time points...but it's not a bad thing to have this information as well to know what proportion of any increases in titer seen in the immunized group are due to boosting versus age-dependent increases in nonspecific binding/background.
Option 2: If it's available, buy "normal" (un-immunized) or SPF chicken serum from a reagent vendor.
Option 3: If you know definitely that the place where you are sourcing the birds from is free of the pathogen of interest you could go and bleed a few birds and pool the sera as the negative control stock (IIRC this is what I did during my MS cos the antigen of interest was influenza H5 haemagglutinin so we were pretty sure the university's flock hadn't been exposed).
Thanks for the reply. On the other hand, Is the cut off value varies between the different secondary antibodies tested as well? for eg between IgY and IgA, is that the same serum tested have a different cut off value?
Yes, so for instance if you test the same bunch of samples and negative control for IgY and IgA, the cutoff should be based on the OD of the negative control of each plate respectively and separately.
The ELISA cut-off is frequently calculated based on results obtained from a population of otherwise normal subjects (“healthy controls”). A cut-off is calculated by adding 2 or 3 SDs on top of the mean OD of the blanc, or antigen-negative wells
t think taht the Sandip answer is the correct. You could test an representative number of healthy control for a representative statistical number and calculate Standard Deviation.
I agree with both of you on the maths. The calculation for an endpoint ELISA is pretty conventional and straightforward - I interpreted Sue Kim's question as being more about what source of serum to use as the negative control.
Thanks Sandip and Antonio ... I am calculating the cut off by using the Mean + 3 Standard deviation. Thanks Shi-Hsia also on clarifying which serum is more suitable as the negative control.
All the responses are really helpful .
Sorry but another question.... is there any source of paper that I can cite based on the calculation of cut-off as below:
Cut off = mean + 3 standard deviation ?
Thanks 🥺
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