title: Comparison of three quantification methods for the TZM-bl pseudovirus assay for screening of anti-HIV-1 agents.
abstract: The TZM-bl pseudovirus assay is commonly used to evaluate the efficacy of neutralizing antibodies and small molecular inhibitors in HIV-1 research. Here, to determine the optimal measurement method for screening anti-HIV-1 inhibitors, we compared three measurement methods based on firefly luciferase and β-galactosidase activities. The 50% tissue culture infective doses (TCID50) of the pseudoviruses were determined using the luciferase, β-galactosidase colorimetric, and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining assays. Three commercial reverse-transcriptase inhibitors (azidothymidine, nevirapine, and lamivudine) were tested as reference drugs to compare the reproducibility, linear correlation, and half maximal inhibitory concentration (IC50) values determined using these methods. In the TCID50 assay, the sensitivity of β-galactosidase colorimetric assay was almost 562 times lower than that of the other two methods. Reproducible dose-response curves were obtained for the inhibitors with all methods; the IC50 values of the inhibitors were not significantly different. Linear regression analysis showed linear correlation between methods. Compared to the β-galactosidase colorimetric assay, the other two methods have the advantage of high sensitivity and are less affected by interference. In conclusion, the luciferase and X-gal staining assays, which can be applied either alone or combined, are recommended for anti-HIV-1 inhibitor screening.
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology, No. 100 Pingleyuan, Beijing 100124, PR China.
authors.forename: Liying
authors.initials: L
authors.lastname: Xing
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology, No. 100 Pingleyuan, Beijing 100124, PR China.
authors.forename: Shunyi
authors.initials: S
authors.lastname: Wang
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology, No. 100 Pingleyuan, Beijing 100124, PR China. Electronic address: hq07616@bjut.edu.cn.
authors.forename: Qin
authors.initials: Q
authors.lastname: Hu
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology, No. 100 Pingleyuan, Beijing 100124, PR China.
authors.forename: Jingtao
authors.initials: J
authors.lastname: Li
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology, No. 100 Pingleyuan, Beijing 100124, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zeng
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D017930:Genes, Reporter; D057809:HEK293 Cells; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D019380:Anti-HIV Agents; D001616:beta-Galactosidase
keywords: HIV-1 pseudovirus; Luciferase; TZM-bl; β-galactosidase