Hello, I am doing end point titer evaluation of serum antibody dilutions. As i am not using any standards or any sort of criterial to refer to as endpoint, can i use 3x of Ag Control values as Cutoff for saying End Point Titer. Ex: If my Ag Control are 0.02 and my serum dilutions are 0.20 OD readings, can i say that titer is above 1:1,00,000 by end point method.
In your case I would recommend to use your highest dilution of your serum antibody that will give you absorbance at least 0.2 or higher. That is going to be your titer.
Thanks. I had 0.27 OD at 10,00,000 dilution and the Ag-Control was 0.02. Hope i can say that titer is above 1,00,000 in end point method
Thanks again
I am confused. Have you done more than a 1 in 1,000,000 dilution? Did it get below 0.02? If your sample values are all higher than your negative serum sample then you have not diluted out far enough to the 'endpoint' (ie you cant say that you have diluted to the point that there is no Ab left in the sample). You need to repeat the assay with more dilutions.
I usually do a number of negative samples (diluted a bit, like 1:100) and I calculate the endpoint cutoff as the mean absorbance of the negative samples plus 2 standard deviations. Just doing 3x negative value doesn't really work to be comparable between ELISAS - if you over or under develop on a different day you cant say the relationship between Abs and Ab will be the same (ie different slope on the curves) so 3x negative for one assay might be low or it could be really high for another assay. Theoretically, if you are using diluted negatives for your cutoff at 1:100 then their endpoint becomes 100 rather than zero, so keep that in mind.
If you need a result soon, just as an indicator and not to publish, and you cannot calculate endpoint then read off OD at a specific value for all your samples (eg 1 in 50,000 dilution) - that should give you a relative idea, but it is only comparable within the one assay.
@Natalie Emilia Stevens . As the animal was boostered 6 times, I believe titer has gone up to the dilution of 1,000,000. Ag-Negative (1:1000 dilution) was 0.02 with least %CV variation from 3 replications and 1,000,000 dilution also had 0.27 OD. Though the ELISA may not perfectly repeatable at this dilutions, still the readings suffice the presence of Ab in 1,000,000 dilutions. Hope i am not wrong in my assumptions.
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