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....

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