Hi everyone,

I have a few questions about using lentiviral plasmids to create pseudoviruses and hope to get some expert advice.

Experimental Setup:

• Packaging plasmid: pCMVdR8.2
• Transfer plasmid: pLenti-CAG-nanoluc
• Envelope plasmid: pcDNA containing Flu H1, N1 genes

After transfecting these plasmids into 293T cells, I collected the supernatant after 3 days. I then incubated the supernatant (sup) with TPCK-trypsin for 1 hour, added FBS to a final concentration of 20%, and then aliquoted and stored the pseudoviruses at -80°C.

To check for infectivity:

I performed a 2-fold serial titration of the influenza pseudoviruses (Flu PSVs) in DMEM supplemented with 10% FBS. I added these serially diluted Flu PSVs to MDCK cells and measured the nanoluciferase signal using Promega NanoGlo. The signals were consistently very high (10^6 to 10^7 RLU), and I observed a clear signal gradient.

The Problem:

However, when I proceeded to a neutralization assay, the results were not successful. I used a known influenza antibody (e.g., CT149) from other literature, which was purified in-house and confirmed to bind to H1 via ELISA.

My neutralization protocol was as follows:

After 3 days, I measured the nanoluciferase signals. I could not observe any neutralization, and the signals showed no clear pattern. The signals were also inconsistent between duplicates and triplicates.

What could be the reason that neutralization is not working? I've checked other literature, but they all used wild-type Cal'09 viruses.

Thanks for all the help!