RAPID AND AUTOMATED TETRAZOLIUM-BASED COLORIMETRIC ASSAY FOR THE DETECTION OF ANTI-HIV COMPOUNDS
RAPID AND AUTOMATED TETRAZOLIUM-BASED COLORIMETRIC ASSAY FOR THE DETECTION OF ANTI-HIV COMPOUNDS
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DOI：
10.1016/0166-0934(88)90134-6
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发表时间：
1988-08-01
影响因子：
3.1
通讯作者：
DECLERCQ, E
中科院分区：
文献类型：
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作者：
PAUWELS, R;BALZARINI, J;DECLERCQ, E
建立了一种快速、灵敏、自动化的检测方法，用于抗HIV药物的体外进化。一个HTLV-I转化了T4细胞系MT-4，这是先前由Koyanagi等人证明的。(1985)对艾滋病毒感染高度敏感并允许感染，作为目标细胞系。以抑制HIV诱导的细胞病变效应为终点。通过四甲基偶氮唑盐(3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltetrazolium)的原位还原比色法，用分光光度法评估艾滋病毒感染细胞和模拟感染细胞的活性。对程序进行了优化，以最大限度地利用多通道吸管、微处理器控制的点胶和光密度读数。模拟感染对照与HIV感染样本的吸光度比约为20。这使得能够准确地测定50%的有效剂量，如3‘’-叠氮-2‘’-二脱氧胸苷(AZT)、2‘’，3‘’-二脱氧胞苷(DdCyd)、硫酸葡聚糖和肝素。与台盼蓝排除法相比，该技术显著减少了劳动时间，并允许对大量化合物的抗HIV活性进行评估。
A rapid, sensitive and automated assay procedure was developed for the in vitro evolution of anti-HIV agents. An HTLV-I transformed T4-cell line, MT-4, which was previously shown by Koyanagi et al. (1985) to be highly susceptible to, and permissive for, HIV infection, served as the target cell line. Inhibition of the HIV-induced cytopathic effect was used as the end point. The viabilty of both HIV-and mock-infected cells was assessed spectrophotometrically via the in situ reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The procedure was optimized as to make optimal use of multichannel pipettes, microprocessor-controlled dispensing and optical density reading. The absorbance ratio of the mock-infected control to the HIV-infected samples was about 20. This allowed an accurate determination of the 50% effective doses, as demonstrated for 3''-azido-2''3''-dideoxythymidine (AZT), 2'',3''-dideoxycytidine (ddCyd), dextran sulfate and heparin. The technique significantly reduced labor time as compared to the trypan blue exclusion method, and permits the evaluation of large numbers of compounds for their anti-HIV activity.