The purification of poliomyelitis virus as studied by complement fixation.
The purification of poliomyelitis virus as studied by complement fixation.
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DOI：
10.4049/jimmunol.78.6.435
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发表时间：
1957-06
影响因子：
4.4
通讯作者：
M. Mayer;H. Rapp;B. Roizman;S. Klein;K. Cowan;David Lukens;C. Schwerdt;F. L. Schaffer;J. Charney
中科院分区：
文献类型：
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作者：
M. Mayer;H. Rapp;B. Roizman;S. Klein;K. Cowan;David Lukens;C. Schwerdt;F. L. Schaffer;J. Charney
已发现脊髓灰质炎病毒和猴肾抗原的补体结合（CF）测定可用于指导和控制脊髓灰质炎病毒纯化所需的浓缩和分级过程。结合感染性测量以及氮分析，CF滴度提供了产量和纯度的信息。已经研究了基本纯化程序的三种变化，以获得产率和纯度。通常，纯化的病毒的产率等于或优于50%，相对于起始材料，纯度增加高达约1000倍。发现用作起始材料的感染的组织培养液（ITCF）含有约0.01至0.2 μg病毒氮/ml。来自切碎的组织培养物的ITCF含有约20 μg/ml的总不可透析N，而来自胰蛋白酶化类型的培养物的ITCF仅含有约3-5 μg的总不可透析N。同时，前一种类型的ITCF显示CF的猴肾抗原滴度比后一种类型高约40倍。初步估计表明，最高度纯化的组分主要由病毒物质组成。来自切碎组织类型ITCF的那些仍含有可观量的猴肾物质，约为25%。另一方面，来自胰蛋白酶化类型的ITCF的纯化产物不含可检测的猴肾抗原，但不能排除存在非抗原性污染物。然而，在猴肾培养物中检测到的感染性病毒仅占实际质量的一小部分，略低于0.1%。其余部分可能代表非感染性病毒或病毒产物，其具有免疫反应性，但在组织培养物上不形成噬斑。通过在蔗糖密度梯度（SDG）中的超离心分离纯化产物的分级导致快速沉降的感染性单位与较慢的非感染性组分分离。两者在脊髓灰质炎抗血清CF试验中均呈反应性。结果表明，感染性滴度不应用作病毒物质的量度，而应采用CF滴度。
Complement fixation (CF) assays for poliomyelitis virus and for monkey kidney antigens have been found useful in guiding and controlling the processes of concentration and fractionation required for the purification of poliomyelitis virus. In conjunction with infectivity measurements, as well as nitrogen analyses, CF titers furnish information on yield and purity. Three variations of the basic purification procedure have been studied with respect to yield and purity gain. Usually, the yield of purified virus was equal to or better than 50%, with purity gains up to about 1000-fold relative to the starting material. The infected tissue culture fluids (ITCF) used as starting material were found to contain about 0.01 to 0.2 μg of virus nitrogen/ml. ITCF from minced tissue cultures contained about 20 μg of total non-dialyzable N per ml, while ITCF from cultures of the trypsinized type contained only about 3–5 μg of total non-dialyzable N. Concomitantly, the former type of ITCF showed monkey kidney antigen titers by CF about 40 times greater than those of the latter type. Tentative estimates indicate that the most highly purified fractions were composed primarily of viral substance. Those which were derived from ITCF of the minced tissue type still contained appreciable amounts of monkey kidney substance, perhaps 25%. On the other hand, purified products from ITCF of the trypsinized type were free of detectable monkey kidney antigens, but the presence of non-antigenic contaminants could not be excluded. However, only a small proportion, somewhat less than 0.1%, of the actual mass was infectious virus, as detected on monkey kidney cultures. The remainder, presumably represents non-infectious virus, or viral products, which are immunologically reactive but which do not form plaques on tissue cultures. Fractionation of purified products by ultracentrifugation in a sucrose density gradient (SDG) led to a separation of fast sedimenting infectious units from slower non-infectious components. Both were reactive in CF tests with polio antisera. It is shown that infectivity titers should not be used as a measure of viral substance, but, instead, CF titers should be employed.