The Lenti-X p24 Rapid Titer Kit uses a standard ELISA to quickly determine the titer of any HIV-1-based lentiviral supernatant. The wells of the included microtiter plate (12 x 8-well strips) are coated with anti-p24 capture antibody, which quantitatively binds the p24 virus core/capsid protein in your test samples. Specifically bound p24 is detected in a typical sandwich ELISA format using a biotinylated anti-p24 secondary antibody, a streptavidin-HRP conjugate, and a color-producing substrate. A p24 standard curve quantifies p24 content, which allows the lentivirus titer to be calculated.

Figure 1. The Lenti-X p24 Rapid Titer Kit protocol. Lentiviral p24 in your packaging cell supernatant is bound to anti-p24-coated wells and detected using a combination of biotinylated anti-p24 secondary antibody, streptavidin-HRP, and a color reagent.

What is p24?

Recombinant lentiviruses generated for research use, whether they are 3rd- or 4th-generation (e.g., generated with Lenti-X kits), contain major structural proteins, including the VSV-G or ecotropic envelope proteins, matrix proteins, and virus core proteins (Figure 2). The gag gene encodes the viral capsid protein (p24), the nucleocapsid proteins (p6 and p7), and a matrix protein (p17). The Lenti-X p24 Rapid Titer Kit specifically measures the amount of p24 capsid protein present in your viral supernatant. The level of p24 correlates directly with virus titer.

Figure 2. p24 is located in the lentiviral capsid and is one of four proteins encoded by the HIV-1 gag gene.

How does it work?

The virus in your lentiviral supernatant is first lysed in the supplied buffer system to release p24. The sample is then applied to a 96-well microtitration plate (made up of 12 separable 8-well strips), in which the wells are precoated with murine anti-p24 capture antibody. After washing to remove unbound lysate, the amount of specifically bound p24 is then detected using a biotinylated anti-p24 secondary antibody, streptavidin-HRP (horseradish peroxidase), and a color development reagent. The kit includes a p24 control which is used to generate a standard curve and calibrate the p24 equivalent of your supernatant and its titer.

Calculating your titer

The titer of your supernatant is determined by correlating its p24 value to virus content or infectivity (Figure 3). Once the relationship is defined, your virus production system can be calibrated to determine a relationship between p24 levels and infectivity. Alternatively, you can calculate the amount of virus based on p24 content as described in the user manual.

As a leader in lentiviral technology, we offer a full range of Lenti-X expression systems and accessories for the effective delivery of cDNAs or shRNAs into any cell type using high-titer lentiviral vectors.

Figure 3. Lentivirus titer correlates with p24 content. A Lenti-X supernatant generated using the pLVX-ZsGreen1 vector was assayed for p24 content using the Lenti-X p24 Titration Kit. HT1080 cells were infected with supernatant dilutions containing the indicated amounts of p24. After 48 hours, transduced cells were counted and mean fluorescence intensity was determined using flow cytometry.

The Lenti-X p24 Rapid Titer Kit provides a simple, rapid method for determining the titer of lentiviral supernatants by using a standard ELISA to measure the amount of the p24 virus core/capsid protein they contain. Lentiviral supernatant samples are added to the wells of a 96-well microtitration plate (made up of 12 separable 8-well strips), which have been coated with an anti-p24 capture antibody which quantitatively binds p24. The bound p24 is detected using a combination of biotinylated anti-p24 secondary antibody, streptavidin-HRP, and a color development reagent. A standard curve is used to correlate p24 values with virus content or infectivity, allowing the amount of virus to be calculated based on p24 content.