Quantification of adeno-associated virus particles and empty capsids by optical density measurement
Quantification of adeno-associated virus particles and empty capsids by optical density measurement
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DOI：
10.1016/s1525-0016(02)00019-9
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发表时间：
2003-01-01
影响因子：
12.4
通讯作者：
Wright, JF
中科院分区：
文献类型：
--
作者：
Sommer, JM;Smith, PH;Wright, JF
我们在这里表明，变性腺相关病毒（AAV）载体的紫外吸光度提供了一个简单，快速，直接的方法定量载体基因组和衣壳蛋白在溶液中。我们测定了衣壳蛋白在260 nm和280 nm处的摩尔消光系数分别为3.72 × 10（6）M-1 cm（-1）和6.61 × 10（6）M-1 cm（-1）。对于重组AAV载体，可以通过包括载体DNA的预测吸光度来计算消光系数。由于纯化的载体制剂中空衣壳的量以可预测的方式降低A（260）/A（280）比率，因此纯化的AAV载体制剂中的载体基因组（vg）和衣壳颗粒（cp）滴度可以由260 nm处的吸光度和A（260）/A（280）比率计算。为了验证该方法，将通过UV吸光度计算的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测定的滴度一致。通过A（260）/A（280）方法测定的cp/vg比率也与通过AAV衣壳ELISA结合Q-PCR测定的那些良好相关。这种新方法提供了一种简单而快速的手段来确定AAV vg滴度和纯化病毒制剂中空颗粒与满颗粒的比率。
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.