Quantification of AAV Particle Titers by Infrared Fluorescence Scanning of Coomassie-Stained Sodium Dodecyl Sulfate–Polyacrylamide Gels
- 1 June 2012
- journal article
- research article
- Published by Mary Ann Liebert Inc in Human Gene Therapy Methods
- Vol. 23 (3), 198-203
- https://doi.org/10.1089/hgtb.2012.049
Abstract
Adeno-associated virus (AAV)-based vectors have gained increasing attention as gene delivery vehicles in basic and preclinical studies as well as in human gene therapy trials. Especially for the latter two—for both safety and therapeutic efficacy reasons—a detailed characterization of all relevant parameters of the vector preparation is essential. Two important parameters that are routinely used to analyze recombinant AAV vectors are (1) the titer of viral particles containing a (recombinant) viral genome and (2) the purity of the vector preparation, most commonly assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by silver staining. An important, third parameter, the titer of total viral particles, that is, the combined titer of both genome-containing and empty viral capsids, is rarely determined. Here, we describe a simple and inexpensive method that allows the simultaneous assessment of both vector purity and the determination of the total viral particle titer. This method, which was validated by comparison with established methods to determine viral particle titers, is based on the fact that Coomassie Brilliant Blue, when bound to proteins, fluoresces in the infrared spectrum. Viral samples are separated by SDS–PAGE followed by Coomassie Brilliant Blue staining and gel analysis with an infrared laser-scanning device. In combination with a protein standard, our method allows the rapid and accurate determination of viral particle titers simultaneously with the assessment of vector purity.Keywords
This publication has 25 references indexed in Scilit:
- The AAV Vector Toolkit: Poised at the Clinical CrossroadsMolecular Therapy, 2012
- Systemic Errors in Quantitative Polymerase Chain Reaction Titration of Self-Complementary Adeno-Associated Viral Vectors and Improved Alternative MethodsHuman Gene Therapy Methods, 2012
- Neutralizing Antibodies Against AAV Serotypes 1, 2, 6, and 9 in Sera of Commonly Used Animal ModelsMolecular Therapy, 2012
- Adenovirus-Associated Virus Vector–Mediated Gene Transfer in Hemophilia BNew England Journal of Medicine, 2011
- Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard MaterialHuman Gene Therapy, 2010
- Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectorsGene Therapy, 2010
- Manufacturing and characterizing AAV-based vectors for use in clinical studiesGene Therapy, 2008
- Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2Gene Therapy, 1999
- Recombinant adeno-associated virus purification using novel methods improves infectious titer and yieldGene Therapy, 1999
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970