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&#160…

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

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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).

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