Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Department of Cell Biology, National Institutes for Food and Drug Control (NIFDC), Beijing, China.
J Virol Methods. 2012 Nov;185(2):267-75. doi: 10.1016/j.jviromet.2012.07.011. Epub 2012 Jul 13.
Among the neutralizing antibody evaluation assays, the single-cycle pseudovirus infection assay is high-throughput and can provide rapid, sensitive and reproducible measurements after a single cycle of infection. Cell counts, pseudovirus inoculation levels, amount of diethylaminoethyl-dextran (DEAE-dextran), and the nonspecific effects of serum and plasma were tested to identify the optimal conditions for a neutralizing antibody assay based on pseudoviruses. Optimal conditions for cell counts, pseudovirus inoculation, and amount of DEAE-dextran were 1 × 10(4)cells/well, 200TCID(50)/well, and 15 μg/ml, respectively. Compared with serum samples, high-concentration anticoagulants reduced the relative light unit (RLU) value. The RLU value increased sharply initially but then decreased slowly with dilution of the plasma sample. Test kits containing 10 HIV-1 CRF07/08_BC pseudovirus strains and 10 plasma samples from individuals infected with HIV-1 CRF07/08_BC were assembled into two packages and distributed to nine laboratories with a standard operating procedure included. For the 10 laboratories that evaluated the test, 17 of 44 (37%) laboratory pairs were considered equivalent. A statistical qualification rule was developed based on the testing results from 5 experienced laboratories, where a laboratory qualified if at least 83% of values lied within the acceptable range.
在中和抗体评价检测方法中，单周期假病毒感染检测方法高通量，在单次感染周期后可提供快速、灵敏和可重现的测量结果。通过细胞计数、假病毒接种水平、二乙氨基乙基葡聚糖（DEAE-dextran）用量以及血清和血浆的非特异性影响测试，确定了基于假病毒的中和抗体检测的最佳条件。细胞计数、假病毒接种和 DEAE-dextran 用量的最佳条件分别为 1×10(4)个细胞/孔、200TCID(50)/孔和 15μg/ml。与血清样本相比，高浓度抗凝剂降低了相对光单位（RLU）值。随着血浆样本稀释，RLU 值最初急剧增加，但随后缓慢下降。包含 10 株 HIV-1 CRF07/08_BC 假病毒株和 10 份 HIV-1 CRF07/08_BC 感染者血浆样本的检测试剂盒被组装成两个包装，并附有标准操作程序分发给 9 个实验室。对于评估该检测的 10 个实验室，44 对实验室中有 17 对（37%）被认为等效。根据 5 个经验丰富的实验室的检测结果制定了一个统计资格规则，如果至少 83%的数值在可接受范围内，则实验室合格。
