Cell-Based AAV Neutralizing Antibody Assay: A Colorimetric Technique to Detect Neutralizing Antibodies Against Specific Adeno-Associated Viruses in Serum

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…

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