Assessing Lentiviral Titer Using an Enzyme-Linked Immunosorbent Assay

Abstract

Source: Vijayraghavan, S., et al. Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells. J. Vis. Exp. (2017).

This video demonstrates the use of a p24 ELISA to estimate viral titers of lentiviral vectors. The assay involves lysing viral particles to release p24, capturing it with specific antibodies, and detecting it with a peroxidase-linked system that produces a measurable color change. By comparing sample absorbance values to a standard curve, the viral titer is quantified.

Protocol

1. Estimation of Viral Titers

1. p24 (Human Immunodeficiency Virus (HIV)-1 p24 capsid protein) -enzyme-linked immunosorbent assay (ELISA) methodNOTE: The assay is carried out using high-binding 96-well plates as per the instructions of the NIH AIDS Vaccine Program for HIV-1 p24 Antigen Capture Assay Kit (see Table of Materials) with modifications.
   1. The next day, wash the wells three times with 200 µL 0.05% Tween 20 in cold PBS (PBS-T solution). Coat the plate with 100 µL of monoclonal anti-p24 antibody at a dilution of 1:1500 in 1x PBS and incubate overnight at 4 °C.
   2. To eliminate non-specific binding, block the plate with 200 µL 1% BSA in PBS; wash three times with 200 µL 0.05% Tween 20 in cold PBS (PBS-T solution) for at least 1 h at room temperature.
   3. Prepare samples: For concentrated vector preparations, dilute 1 µL of the sample 100-fold by adding 89 µL of dd-H20 and 10 µL of Triton X-100 (final concentration of 10%). For non-concentrated preparations, prepare ten-fold diluted samples (add 80 µL of dd-H20 and 10 µL of Triton X-100 (final concentration of 10%) to 10 µL of the sample).NOTE: Samples can be stored at -20 °C at this step for an extended period of time for later usage.
   4. Prepare HIV-1 standards by applying a 2-fold serial dilution (with a starting concentration 5 ng/mL).
   5. Dilute concentrated samples (from 1:100 pre-diluted stocks) in RPMI 1640 (Roswell Park Memorial Institute 1640 medium) supplemented with 0.2% Tween 20 and 1% BSA to establish 1:10,000, 1:50,000, and 1:250,000 dilutions. Dilute non-concentrated samples (from 1:10 pre-diluted stocks) in RPMI 1640 supplemented with 0.2% Tween 20 and 1% BSA to establish 1:500, 1:2500, and 1:12,500 dilutions.
   6. Apply samples on the plate in triplicates and incubate overnight at 4 °C.
   7. The next day, wash the wells six times and incubate at 37 °C for 4 h with 100 µL polyclonal rabbit anti-p24 antibody, diluted 1:1000 in RPMI 1640, 10% FBS, 0.25% BSA, and 2% normal mouse serum (NMS).
   8. Wash six times as above and incubate at 37 °C for 1 h with goat anti-rabbit horseradish peroxidase IgG diluted 1:10,000 in RPMI 1640 supplemented with 5% normal goat serum, 2% NMS, 0.25% BSA, and 0.01% Tween 20.
   9. Wash the plate, as above, and incubate with TMB (3,3′,5,5′-Tetramethylbenzidine) peroxidase substrate at room temperature for 15 min.
   10. Stop the reaction by adding 100 µL of 1 N HCL. Measure sample absorbance at 450 nm using the absorbance plate reader.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
p24 ELISA reagents
Monoclonal anti-p24 antibody	NIH AIDS Research and Reference Reagent Program	3537
HIV-1 standards	NIH AIDS Research and Reference Reagent Program	SP968F
Polyclonal rabbit anti-p24 antibody	NIH AIDS Research and Reference Reagent Program	SP451T
Goat anti-rabbit horseradish peroxidase IgG	Sigma Aldrich	12-348	Working concentration 1:1500
Normal mouse serum, Sterile, 500mL	Equitech-Bio	SM30-0500
Goat serum, Sterile, 10mL	Sigma	G9023	Working concentration 1:1000
TMB peroxidase substrate	KPL	5120-0076	Working concentration 1:10,000