Neutralization Assay: Comparision of IC50 values between different viral loads

Question

I've been performing Neutralization assays for HIV pseudovirus against a dilution series of different antibodies, and was wondering whether the viral load influences the measured IC50.

My specific reasoning: I apparently understand the concept of normalization (0-100% neutralization). But if you calculate the virus titer beforehand (e.g. 1000 RLU/uL), and add exactly 10'000 RLU viral load (10uL virus) to every well, wouldn't you get a different IC50 value for the same virus with the same antibody when you use e.g. 20'000 RLU (20uL virus), because in this scenario you would need more antibody (thus higher IC50) to achieve a 50% neutralization for the 20'000 RLU wells compared to the 10'000 RLU wells.

What I don't understand: Apparently the difference of viral loads can be resolved by normalization, but this I don't understand. Shouldn't you "always" get a lower IC50 value for the same virus with the same antibody dilution series when you add less virus (smaller viral load). Doesn't this mean that you can't compare different IC50 values between different assays and viruses if you don't know the original viral load? (e.g. smaller IC50 values could be explained by smaller viral load instead of higher affinity of the antibody to the virus)

Thank you for clarifying.

Answer 1

Yes. This is one of the basic concepts in virology, and presumably in other branches of biology too. It doesn't just apply to antibodies; it also applies to chemical (drug) treatments. You need to apply a standardized concentration of the virus(es) across your treatments or you can't compare them.

A good example of this is the classic influenza assay HAI (HemAgglutination Inhibition), which is a test of sera for the presence of antibodies inhibiting hemagglutination and therefore response to influenza infection, requires you to standardize the HA (Hemagglutinin) content of your viruses before performing the assay via the HA assay.

For other viruses you might use a different standardization process, perhaps something like a TCID50 (Tissue Culture Infectious Dose 50% endpoint) or a plaque assay used then to work out that X infectious units (in TCID50/ml or pfu/ml respectively) is a good point to work at for your antibody assay.