DETECTION OF HIV BY A MAGNETIC BEAD BASED VIRAL CAPTURE ASSAY

一种从体液中检测艾滋病毒的新技术正在开发中 如血清、血浆和感染细胞的培养上清液。H9 用HIV-MN毒株及其培养上清感染细胞 于第4天采集细胞，测定上清液中的p24抗原。病毒 流体中存在的颗粒通过捕获浓缩 抗体包被的珠子。大小均匀的颗粒主要被包覆 然后与抗p24和抗gp120抗体反应。 分别为免疫球蛋白G类抗体。孵化16年后 在寒冷中数小时，用缓冲液清洗珠子，并将其储存在4℃下。 然后将培养上清液与珠子一起在寒冷中孵化 16个小时。这些珠子被洗了两次，并用胍处理 异硫氰酸盐缓冲液可去除病毒。从被剥离的 制备材料，使用SK 38/39引物和 扩增产物与P-32标记的SK-19探针杂交 液体杂交技术。在我们的初步实验中，病毒粒子 富含涂有抗HIV抗体的磁珠和 放射自显影印迹显示强阳性信号。这种方法 正在适应于检测感染患者血清和血浆中的病毒 个人，以努力获得敏感性和特异性。这 这项技术将用于检测病毒以及表征病毒 直接从珠子中通过聚合酶链式反应测序分离病毒。

A novel technique is being developed for detecting HIV from body fluids such as serum, plasma and culture supernatants of infected cells. H9 cells were infected with HIV-MN strain and the culture supernatants collected on day 4. p24 antigen was measured in the supernatant. Virus particles present in the fluids were concentrated by capture with antibody coated beads. Particles of uniform size were coated primarily with anti IgG antibodies and then reacted with anti p24 and anti gp120 separately, both being IgG class antibodies. After incubation for 16 hours in the cold, the beads were washed with buffer and stored at 4 C. Culture supernatants were then incubated with the beads in the cold for 16 hours. The beads were washed twice and treated with guanidine isothiocyanate buffer to strip off the virus. RNA from the stripped material was prepared and used for RT-PCR using SK 38/ 39 primers and amplified products were hybridized with P-32 labelled SK 19 probe by liquid hybridization technique. In our preliminary experiments, virions were enriched with magnetic beads coated with anti HIV antibodies and showed strong positive signals on an autoradiographic blot. This method is being adapted to detecting virus in the serum and plasma of infected individuals in an effort to derive sensitivity and specificity. This technique will be of use in detecting virus as well as in characterizing virus isolates by PCR sequencing directly from the bead.