Sommer Jürg M, Smith Peter H, Parthasarathy Sumathy, Isaacs Jesse, Vijay Sharmila, Kieran Jane, Powell Sharon K, McClelland Alan, Wright J Fraser
Avigen, Inc., 1301 Harbor Bay Parkway, , Alameda, California 94502, USA.
Mol Ther. 2003 Jan;7(1):122-8. doi: 10.1016/s1525-0016(02)00019-9.
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.
我们在此表明，变性腺相关病毒（AAV）载体的紫外吸光度为定量溶液中的载体基因组和衣壳蛋白提供了一种简单、快速且直接的方法。我们测定衣壳蛋白在260 nm处的摩尔消光系数为3.72×10⁶ M⁻¹ cm⁻¹，在280 nm处为6.61×10⁶ M⁻¹ cm⁻¹。对于重组AAV载体，消光系数可通过纳入载体DNA的预测吸光度来计算。由于纯化载体制剂中空心衣壳的量以可预测的方式降低A₂₆₀/A₂₈₀比值，因此纯化AAV载体制剂中的载体基因组（vg）和衣壳颗粒（cp）滴度可根据260 nm处的吸光度和A₂₆₀/A₂₈₀比值来计算。为验证该方法，将通过紫外吸光度计算得到的vg和cp滴度与通过定量（Q）-PCR和衣壳ELISA测定的滴度进行比较。通过吸光度测定的vg滴度与通过Q-PCR测定的滴度吻合良好。通过A₂₆₀/A₂₈₀方法测定的cp/vg比值也与通过AAV衣壳ELISA结合Q-PCR测定的比值高度相关。这种新方法为确定纯化病毒制剂中AAV vg滴度以及空心与完整颗粒的比例提供了一种简单快速的手段。
Zotero 插件