The Virus-Like Particles Capture Assay: A Method to Isolate Antigen-Displaying VLPs from a Sample Using Neutralizing Antibody-Conjugated Magnetic Beads

Neutralizing antibodies, or NAbs, are secreted by cells to protect them against invading viruses.

To study the interactions of NAbs with viruses, use engineered virus-like particles, or VLPs. These nanosized structures contain an assembly of virus-derived proteins without viral genetic material, making them non-infectious. VLPs are surrounded by a lipid bilayer embedded with antigenic envelope glycoproteins, which present a neutralizing epitope, a specialized region to which the NAbs bind.

Add the VLPs to a tube containing neutralizing antibodies bound to protein-conjugated magnetic beads. Incubate under agitation to prevent the beads from settling down. During incubation, the epitope region of the VLPs recognizes the complementary paratope on the Fab region of the NAbs, resulting in antigen-antibody complexes. This, in turn, helps the magnetic beads to capture the VLPs.

Place the tube in a separation rack equipped with a strong magnet. Under the influence of a magnetic field, the beads complexed with VLPs move toward the magnet without disturbing the antigen-antibody interaction. This step captures the bound VLPs, while the ones lacking specific antigens remain in the supernatant.

Discard the supernatant, and wash the pellet with washing buffer to remove unbound VLPs. Resuspend the VLP-bead complexes in an elution buffer, which causes the VLPs to disengage from the beads and appear in the supernatant.

Aspirate the isolated antigen-displaying VLPs into a fresh tube, and store them under refrigeration for further analysis.

Begin by resuspending the magnetic beads by either pipetting up and down or mixing on a rotator at 50 RPM for at least 5 minutes.

Meanwhile, prepare the antibody solution containing the bNAbs. Use 10 micrograms of each bNAb and 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 wall 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 3 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 resuspending 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 1 milliliter, add PBS to adjust the sample volume to 1 milliliter, then, resuspend 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 milliliters 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 SDS-PAGE samples, suspend the beads in 20 to 80 microliters of Laemmli buffer, and incubate at 95 degrees Celsius for 5 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 -20 degrees Celsius.