In my lab, we develop production systems for viral vectors and virus-like particle, or VLP-based vaccines. The VLP capture assay allows for the very sensitive detection of target antigens displayed on VLPs. In this assay, we use broadly-neutralizing antibodies directed against neutralization epitopes to prove the exposure of these epitopes on the VLP surface.
This is an important quality assessment for vaccine candidates, as only VLPs displaying neutralization-sensitive epitopes will be able to elicit a neutralizing antibody response in vaccinees. Begin by suspending the magnetic beads by either pipetting up and down, or mixing on a rotator at 50 RPM for at least five minutes. Meanwhile, prepare the antibody solution containing the bNABs.
Use 10 micrograms of each bNAB in 200 microliters of antibody binding and washing buffer per reaction. For each reaction, transfer 50 microliters of the magnetic bead solution into a 1.5-milliliter reaction tube, then place the tubes on the magnetic separation rack. Wait until the beads gather at the tube ball to ensure that all beads are collected, then remove the supernatant.
Remove the magnet and suspend the beads in 200 microliters of the previously-prepared bNAB solution. Incubate for 30 minutes to three hours while mixing on a rotator at 50 RPM at room temperature. After the incubation, place the reaction tubes in the magnetic separation rack, wait, and remove the supernatant.
Remove the tubes from the magnet and wash the beads by re-suspending in 200 microliters of antibody binding and washing buffer. Repeat the wash with antibody binding and washing buffer, removing as much washing buffer as possible when finished. Add the samples to the bead-bound bNABs.
If the added sample volume is below one milliliter, add PBS to adjust the sample volume to one milliliter, then re-suspend the beads by gently pipetting. Incubate the samples and beads for 2.5 hours on a rotator at room temperature, ensuring that the beads stay in suspension and the solution is thoroughly mixed during incubation. Place the tubes on the magnet and remove the supernatant, then wash the magnetic beads by suspending them in 200 microliters of washing buffer.
Suspend the beads in 100 microliters of washing buffer and transfer the suspension to a clean, heat-resistant reaction tube. Place the tube on the magnetic separation rack and remove the supernatant completely. To prepare denatured STS-PAGE samples, suspend the beads in 20 to 80 microliters of Laemmli buffer and incubate at 95 degrees Celsius for five minutes.
Proceed directly with SDS-PAGE, placing the tubes on a magnetic rack to separate the beads from the solution. Alternatively, store the samples at minus 20 degrees Celsius. A representative outcome of VLPs first captured from cell-free cell culture supernatant and VLP pellets using bNABs, and subsequently subjected to Western blot analysis to detect viral core proteins, is shown here.
The beads used for the capture assay were coated with three different bNABs directed against neutralization epitopes of the envelope glycoproteins and isotype antibodies serving as negative controls. Using isotope antibody-coated beads, no Gag proteins were detectable in VLP samples containing bald VLPs, that is, Env-negative VLPs, or Env-displaying VLPs, demonstrating that the unspecific binding of VLPs to beads coated with human antibodies did not mediate VLP capture. Bald VLPs were also not bound by bNABs-coated beads, and consequently, no Gag proteins were detectable in the Western blot analysis.
In contrast, all three bNABs captured VLPs displaying Env-proteins, therefore, Gag proteins were readily detected, demonstrating the presence of neutralization epitopes in the Env-glycoproteins displayed on VLPs. Crucial to the success of the assay:a thorough mixing of VLPs with antibody-coated beads. This is best achieved using volumes larger than 500 microliters under rotation.
Alternatively, captured VLPs can be eluted under non-reducing conditions. This enables the analysis of VLPs employing immuno-electron microscopy, or the investigation of the displayed antigens using native PAGE.
