Hi All,
I am trying to work out EBV titer using the Reed and Muench calculator so that the mice can be injected with 1000 TD50. To titrate, I infected PBMCs from cord blood with 6 dilutions of the virus (dilution factor:10). After 6 weeks, I saw that 2 out of the total 4 wells were negative for LCL foci in the last dilution (i.e.10^-5) while the rest were all positive. When I feed this numbers in the Reed and Muench calculator from here: https://link.springer.com/protocol/10.1007%2F978-1-59745-394-3_24, it yields a value of 1.00E-04 for TCID50/ml while TCID is 1.00E+05. Could someone explain to me what is going on here and how to work out how much virus to inject to be able to get 1000 TD50 per mouse?
Any help is much appreciated, happy to talk via email as well.
Thanks
Thanks Vladimir, that's very helpful. I should be able to check lower dilutions as well.
2/4 may be 50%, but the number would be more meaningful if you had a larger number of test wells, as well as dilutions higher than what gave you 50%. The method of Reed & Muench was developed before calculators and desk top computers (or laptops) were available. There are good programs for Probit Analysis (e.g., Sigma Stat), which is the proper statistics for such a distribution. In any assay, the endpoint value is insufficient for an answer. You also need the precision (SEM error bars, confidence limits) of that value. The Probit analysis provides that.
When you are close to the 50% value, the Poisson distribution is also helpful, e.g., when 63% get the result and 37% do not, one has an average of one event per number of cells seeded. (P(x,a) = (exp(-a)*a^x)/x!, where a = average number of events and x = number of events measured). So for a=1 and x=0, P(0,1) = exp(-1) * 1/1 = exp(-1) = 0.37.
Hope this helps.