I am trying to optimize Taqman based qRT-PCR and p24 based ELISA to determine the viral titer for HIV-1 in macrophages exposed to HIV-1 BaL. Is there any established protocol for TCID determination?
HI
Depending of your objectives is several ways to do it
1. just a p24 elisa (I recommend the perkin elmer one)
2. qRT-PCR for conserved areas
3. if you need ALU PCR to quantify numbers of copies per cell.
4. See Siliciano's papers for ultra sensitive PCR also
There are several ways, but dependent of your interests
As indicated, there are various methohs. Traditionally, qRT-PCR, but there is also a colorimetric method from Roche and available in Sigma for detrmination of RT activity and its inhibitors. However, perhaps the best is the use of pseudotyped luciferase reporter HIV-1 virus.We use a non-macrophage cell line (TZM-bl) that expresse CCR5 and CD4 but also it is a luciferase cell reporter (thus obviating the need for pseudotyped luciferase reporter virus).
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