The Virus-Like Particles Capture Assay: A Method to Isolate Antigen-Displaying VLPs from a Sample Using Neutralizing Antibody-Conjugated Magnetic Beads
Published: April 30, 2023
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 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. 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). Pellet the producer cells by centrifugation at 100 x …
Representative 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 a defined p55 concentration is prepared. The cell-free culture supernatant consists of a mixture of p55 Gag VLPs, host cell proteins,…
Divulgaciones
The authors have nothing to disclose.
Materials
| 1.5 mL reaction tubes | Eppendorf | ||
| 10x PBS | gibco | 70011044 | |
| Antibodies (bnAbs) | Polymun Scientic | ||
| Dynabeads Protein G Immunoprecipitation Kit |
invitrogen (Thermo Fisher Scientific) | 10007D | includes buffers and washing 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) syringe filters, 0.45 µm |
Carl Roth | KC89.1 | |
| PVDF transfermembrane, 0.45 µm | 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 |
Tags
Citar este artículo
The Virus-Like Particles Capture Assay: A Method to Isolate Antigen-Displaying VLPs from a Sample Using Neutralizing Antibody-Conjugated Magnetic Beads. J. Vis. Exp. (Pending Publication), e21155, doi: (2023).