Quantification of adeno-associated virus particles and empty capsids by optical density measurement.
We show here that UV absorbance of denatured adeno-associated virus (AAV) vector provides a simple, rapid, and direct method for quantifying vector genomes and capsid proteins in solution. We determined the molar extinction coefficients of capsid protein to be 3.72 x 10(6) M(-1) cm(-1) at 260 nm and 6.61 x 10(6) M(-1) cm(-1) at 280 nm. For recombinant AAV vectors, extinction coefficients can be calculated by including the predicted absorbance of the vector DNA. Since the amount of empty capsids in purified vector preparations lowers the A(260)/A(280) ratio in a predictable manner, the vector genome (vg) and capsid particle (cp) titers in purified AAV vector preparations can be calculated from the absorbance at 260 nm and the A(260)/A(280) ratio. To validate this method, the vg and cp titers calculated by UV absorbance were compared with titers determined by quantitative (Q)-PCR and capsid ELISA. The vg titers determined by absorbance agreed well with titers determined by Q-PCR. The cp/vg ratio determined by the A(260)/A(280) method also correlated well with those determined by AAV capsid ELISA in conjunction with Q-PCR. This new method provides a simple and rapid means to determine AAV vg titers and the ratio of empty to full particles in purified virus preparations.
Sommer
Jürg M
JM
Avigen, Inc., 1301 Harbor Bay Parkway, , Alameda, California 94502, USA. jsommer@avigen.com
Smith
Peter H
PH
Parthasarathy
Sumathy
S
Isaacs
Jesse
J
Vijay
Sharmila
S
Kieran
Jane
J
Powell
Sharon K
SK
McClelland
Alan
A
Wright
J Fraser
JF
D002213:Capsid; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics / Q000302:isolation & purification*; D005822:Genetic Vectors; D006801:Humans; D055095:Optics and Photonics; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014771:Virion