Njayou M, Quash G
Laboratoire de Biochimie Microbienne, Faculté des Sciences, Yaounde, Cameroun.
J Virol Methods. 1991 Apr;32(1):67-77. doi: 10.1016/0166-0934(91)90186-4.
The purification of viruses by ultracentrifugation in regions with limited resources is often hampered by problems of instrument maintenance. Viral antigens must therefore be imported, which results in delays in obtaining epidemiological data. To address this problem, we undertook the development of purification procedures by affinity chromatography using measles virus (strain Y15) and monkey IgG (anti-measles) coupled covalently via their carbohydrate residues to glass spheres as a model system. The measles virus prepared in this way was compared with measles virus purified by ultracentrifugation using three criteria: the degree of purification, yield and the sensitivity in HA and ELISA. The purification of the antigen by ultracentrifugation (AgU) was undertaken using 10(9) infected cells, and by affinity chromatography (AgAc) using 10(7) infected cells. The total concentration of proteins before purification was 5000 mg for AgU and 11.3 mg for AgAc. After purification by AgU 21.5 mg protein was obtained which was assessed by specific hemagglutinating units (HAU/mg protein) and found to be 179 for AgU and 372-744 for AgAc. Based on total HAU, the yield by centrifugation was 2.4%, whereas the affinity chromatography produced essentially 100%. By ELISA, using reference sera to calibrate the antigens, maximum OD was obtained with 0.5 microgram/well of AgAc and with 2 micrograms/well of AgU. From the relatively small amount of starting material, good yields were obtained with increased specific activity economy in coating by ELISA. These results favour the adoption of affinity chromatography for purifying viral antigens.
在资源有限的地区，通过超速离心法纯化病毒常常因仪器维护问题而受阻。因此，病毒抗原必须进口，这导致获取流行病学数据出现延迟。为解决这一问题，我们以麻疹病毒（Y15株）和通过其碳水化合物残基与玻璃珠共价偶联的猴IgG（抗麻疹）作为模型系统，开展了亲和层析纯化程序的研发。将以这种方式制备的麻疹病毒与通过超速离心法纯化的麻疹病毒在三个标准下进行比较：纯化程度、产量以及血凝试验（HA）和酶联免疫吸附测定（ELISA）中的灵敏度。通过超速离心法（AgU）纯化抗原使用10⁹个感染细胞，通过亲和层析法（AgAc）纯化使用10⁷个感染细胞。纯化前蛋白质的总浓度对于AgU为5000毫克，对于AgAc为11.3毫克。通过AgU纯化后获得21.5毫克蛋白质，通过特异性血凝单位（HAU/毫克蛋白质）评估，发现AgU为179，AgAc为372 - 744。基于总HAU，离心法的产量为2.4%，而亲和层析法基本上达到100%。通过ELISA，使用参考血清校准抗原，在每孔0.5微克的AgAc和每孔2微克的AgU时获得最大光密度（OD）。从相对少量的起始材料中，通过ELISA包被获得了良好的产量且比活性提高，经济性更好。这些结果支持采用亲和层析法纯化病毒抗原。
