title: Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1.
abstract: 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.
authors.affiliation: Department of Immunology, Duke University Medical Center, Durham, NC, United States; Department of Surgery, Duke University Medical Center, Durham, NC, United States. Electronic address: marcella.sarzottikelsoe@dm.duke.edu.
authors.forename: Marcella
authors.initials: M
authors.lastname: Sarzotti-Kelsoe
authors.affiliation: Vaccine Research Center, National Institutes of Health (NIH), Bethesda, MD, United States.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Bailer
authors.affiliation: Vaccine Research Center, National Institutes of Health (NIH), Bethesda, MD, United States.
authors.forename: Ellen
authors.initials: E
authors.lastname: Turk
authors.affiliation: Vaccine Research Center, National Institutes of Health (NIH), Bethesda, MD, United States.
authors.forename: Chen-li
authors.initials: CL
authors.lastname: Lin
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, United States.
authors.forename: Miroslawa
authors.initials: M
authors.lastname: Bilska
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, United States.
authors.forename: Kelli M
authors.initials: KM
authors.lastname: Greene
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, United States.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Gao
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, United States.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Todd
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, United States.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Ozaki
authors.affiliation: Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Seaman
authors.affiliation: Vaccine Research Center, National Institutes of Health (NIH), Bethesda, MD, United States.
authors.forename: John R
authors.initials: JR
authors.lastname: Mascola
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, United States.
authors.forename: David C
authors.initials: DC
authors.lastname: Montefiori
mesh_terms: D057134:Antibodies, Neutralizing / Q000097:blood*; D057205:Automation, Laboratory / Q000592:standards; D015415:Biomarkers / Q000097:blood; D019983:Guideline Adherence / Q000592:standards; D015483:HIV Antibodies / Q000097:blood*; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000592:standards*; D006801:Humans; D057230:Limit of Detection; D009500:Neutralization Tests / Q000592:standards*; D015588:Observer Variation; D017410:Practice Guidelines as Topic / Q000592:standards; D011237:Predictive Value of Tests; D011786:Quality Control; D015203:Reproducibility of Results; D013997:Time Factors; D014162:Transfection
chemical_list: D057134:Antibodies, Neutralizing; D015415:Biomarkers; D015483:HIV Antibodies
keywords: Assay validation; GCLP; HIV; Neutralizing antibody; TZM-bl cells