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

Abstract

Source: Rosengarten, J. F., et al. Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay. J. Vis. Exp. (2022).

In this video, we demonstrate an immunoprecipitation assay, called the VLP capture assay, to isolate antigen-displaying virus-like particles, or VLPs, from a sample. The antigen binds with specific antibodies bound to magnetic beads, forming an immobilized antigen-antibody complex that is later separated and stored for further analysis.

Protocol

1. Sample preparation

1. Seed the VLP producer suspension cell lines derived from 293-F cells expressing HIV structural genes gag alone or in concert with env at a low cell density of 0.5 x 10⁶ cells per mL in 293-F Expression Medium.
2. Let them expand for 3-4 days at 37 °C and 8% CO₂ in a shaker incubator rotation with an orbit of 5 cm and 135 rounds per minute (rpm).
3. Pellet the producer cells by centrifugation at 100 x g for 5 min. Filter the clarified supernatant to remove residual cells and cell debris using 0.45 µm polyvinylidene fluoride (PVDF) membrane syringe filters to obtai....

Results

Figure 1: Schematic illustration of key steps of the VLP capture assay using cell-free supernatants. (1) The VLP capture assay starts with the binding of the HIV-1 bNAbs to the protein G-coupled magnetic beads. Meanwhile, the VLP solution with .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL reaction tubes	Eppendorf
10x PBS	gibco	70011044
Antibodies (bnAbs)	Polymun Scientic
Dynabeads Protein G<br/> Immunoprecipitation Kit	invitrogen (Thermo Fisher Scientific)	10007D	includes buffers and washing<br/> solutions
FreeStyle 293-F cells	invitrogen (Thermo Fisher Scientific)	R790-07
FreeStyle 293 Expression Medium	invitrogen (Thermo Fisher Scientific)	12338026
Magnetic separation rack	New England Biolabs	S1509S	for 12 x 1.5 mL or 6 x 1.5 mL tubes
Optima XE-90 ultracentrifuge	Beckman Coulter
Polyvinylidene fluoride (PVDF)<br/> syringe filters, 0.45 &micro;m	Carl Roth	KC89.1
PVDF transfermembrane, 0.45 &micro;m	Carl Roth	T830.1
QuickTiter HIV p24 ELISA	Cell Biolabs	VPK-108-H
Rotator	Heidolph	REAX2
SW28 rotor	Beckman Coulter
Thermomixer	Cel Media	basic
Trehalose dihydrate	Carl Roth	8897.2
Ultra Clear centrifuge tubes	Beckman Coulter	344058