Q: Can this kit be used with any lentivirus pseudotype?
A: Our Lentivirus-Associated p24 ELISA Kit uses two chemical polymers (Reagent A and Reagent B) that form a complex with the virus, independent of the envelope protein. These complexes are then pelleted, dissolved, and disrupted to release p24. The antibody used in this kit is specific for lentivirus associated HIV-1 p24 core protein, which is independent of the envelope protein. This kit will work on any lentivirus pseudotype because it is not specific for VSVG lentivirus.
Q: How do I make the standard curve?
A: We use Excel to generate the standard curve and therefore have to use a linear trendline. If the standard curve is not linear or the r2 is low, the upper and lower values of the curve that are outside of the linear portion can be eliminated. The equation of the trendline can be used to calculate the concentrations of unknowns by solving for x in y=mx+b, which is: (OD value-b)/m for linear lines.
Q: How are dilutions accounted for in the calculation?
A: The 0.25 mL / 1 mL in the calculation is based on the volumes used in the assay protocol. The total sample volume used in the assay is 1 mL (step 1) and after spinning down the 1 mL, the pellet is resuspended in 250 µL of sample diluent (step 3). This gets factored into the calculation as 0.25 mL / 1 mL. Any dilution made to the sample would get factored into the equation as the dilution factor. For example, if you used 10 µL of viral supernatant, 990 µL media would be added to bring up the total volume to 1 mL, resulting in a 1:100 dilution. This is then accounted for as a 100 dilution factor.
Q: Can you provide an example calculation?
A: Here is an example calculation assuming the standard curve gave a p24 concentration of 50 ng/mL:
50 ng/mL p24 (from standard curve) x 100 (dilution factor) x 0.25 mL/1.0 mL = 1250 ng/mL p24
1250ng/mL x 1.25x10^7 LPs/ng p24 (based on p24 MW) = 1.56 x 10^10 LP/mL
1.56 x 10^10 LP/mL x 1 TU/100-1000 LP = 1.56 x 10^7-8 TU/mL