Cell-Based AAV Neutralizing Antibody Assay: A Colorimetric Technique to Detect Neutralizing Antibodies Against Specific Adeno-Associated Viruses in Serum
Published: April 30, 2023
Abstract
Source: Bass-Stringer, S., et al. A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay. J. Vis. Exp. (2021)
In this video, we demonstrate an in vitro colorimetric assay to detect the presence of neutralizing antibodies against alkaline phosphatase-expressing recombinant adeno-associated virus serotype 6 (rAAV6) vectors in sheep serum samples. The assay utilizes the reaction between the AAV encoding a human placental alkaline phosphatase gene and its substrate to generate an insoluble purple product.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. A schematic overview of the assay protocol is provided in Figure 1. 1. Initial preparation For assessment in sheep: collect blood in 8 mL serum separator clot activator tubes (see Table of Materials), leave the blood sample at room temperature (RT) for 20-30 min, and subsequentl…
Representative Results
Figure 1: Schematic diagram of NAb assay protocol. (A) Visual representation of the NAb assay illustrating the primary steps involved in the three-day protocol. Briefly, cells are grown and plated overnight. The following day, serial dilutions of serum are prepared, incubated with AAV, and then incubated with the cells overnight. The next day, cells are fixed, washed, incubated, combined with the…
Offenlegungen
The authors have nothing to disclose.
Materials
| 0.05% Trypsin/EDTA | Gibco | 25300-054 | |
| 50 mL conical centrifuge tube | Falcon | 14-432-22 | Or equivalent |
| 75 cm2 square flasks | Falcon | 353136 | Or equivalent |
| 96 well flat bottomed plate | Falcon | 353072 | |
| AAV6-CMV-hPLAP Vector | Muscle Research & Therapeutics Lab (University of Melbourne, Australia) AAV6-CMV-hPLAP can be provided upon request. | ||
| Aluminium foil | |||
| Anti-AAV6 (intact particle) mouse monoclonal antibody, (ADK6) | PROGEN | 610159 | Positive control monoclonal antibody |
| BCIP/NBT | SIGMAFAST | B5655 | |
| Cell and tissue culture safety cabinet | |||
| Electronic Pipette | 5 & 10 mL stripette inserts | ||
| Fetal Bovine Serum | Gibco | 10099-141 | |
| Haemocytometer | |||
| High glucose Dulbecco's Modified Eagle Medium (DMEM) | Gibco | 11965118 | |
| HT1080 cells | ATCC | ||
| Incubator | 37 °C, 5% CO2 | ||
| Light microscope with camera | Capable of taking photos with a 4x objective lens | ||
| Oven | For a 65 °C incubation | ||
| Paraformaldehyde | MERCK | 30525-89-4 | |
| Water bath | |||
| Penicillin Streptomycin | Gibco | 15140-122 |
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Cell-Based AAV Neutralizing Antibody Assay: A Colorimetric Technique to Detect Neutralizing Antibodies Against Specific Adeno-Associated Viruses in Serum. J. Vis. Exp. (Pending Publication), e21227, doi: (2023).