Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1

• Published in: Journal of immunological methods Vol. 409; pp. 131 - 146
• Main Authors: Sarzotti-Kelsoe, Marcella, Bailer, Robert T., Turk, Ellen, Lin, Chen-li, Bilska, Miroslawa, Greene, Kelli M., Gao, Hongmei, Todd, Christopher A., Ozaki, Daniel A., Seaman, Michael S., Mascola, John R., Montefiori, David C.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2014
• Subjects: Antibodies, Neutralizing - blood; Assay validation; Automation, Laboratory - standards; Biomarkers - blood; clinical trials; compliance; detection limit; GCLP; gene expression; Guideline Adherence - standards; guidelines; HeLa Cells; High-Throughput Screening Assays - standards; HIV; HIV Antibodies - blood; HIV Infections - blood; HIV Infections - diagnosis; HIV Infections - immunology; HIV-1 - genetics; HIV-1 - immunology; Human immunodeficiency virus 1; Humans; Limit of Detection; luciferase; neutralization; neutralization tests; Neutralization Tests - standards; neutralizing antibodies; Neutralizing antibody; Observer Variation; Practice Guidelines as Topic - standards; Predictive Value of Tests; Quality Control; reporter genes; Reproducibility of Results; Time Factors; Transfection; TZM-bl cells; viruses; TZM-bl cells; GCLP; HIV; Assay validation; Neutralizing antibody
• ISSN: 0022-1759; 1872-7905; 1872-7905
• DOI: 10.1016/j.jim.2013.11.022

The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of pre-clinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format.